Micromass Quattro API Operators Guide

Waters Quattro micro
API Mass Spectrometer

Operator’s Guide
71500058602/Revision D
Copyright © Waters Corporation 2003−2010

All rights reserved
Copyright notice
© 2003−2010 WATERS CORPORATION. PRINTED IN THE UNITED
STATES OF AMERICA AND IN IRELAND. ALL RIGHTS RESERVED. THIS
DOCUMENT OR PARTS THEREOF MAY NOT BE REPRODUCED IN ANY
FORM WITHOUT THE WRITTEN PERMISSION OF THE PUBLISHER.
The information in this document is subject to change without notice and
should not be construed as a commitment by Waters Corporation. Waters
Corporation assumes no responsibility for any errors that may appear in this
document. This document is believed to be complete and accurate at the time
of publication. In no event shall Waters Corporation be liable for incidental or
consequential damages in connection with, or arising from, its use.
Trademarks
Waters and Micromass are registered trademarks of Waters Corporation, and
MassLynx, Quattro, and “THE SCIENCE OF WHAT’S POSSIBLE.” are
trademarks of Waters Corporation.
PEEK is a trademark of Victrex Corporation.
Rheodyne is a registered trademark of Rheodyne, L.P.
Swagelok is a registered trademark of Swagelok Inc.
Upchurch is a registered trademark of Scivex, Inc.
Windows is a registered trademark of Microsoft Corporation.
Other registered trademarks or trademarks are the sole property of their
owners.
ii
Customer comments
Waters’ Technical Communications department invites you to tell us of any
errors you encounter in this document or to suggest ideas for otherwise
improving it. Please help us better understand what you expect from our
documentation so that we can continuously improve its accuracy and
usability.
We seriously consider every customer comment we receive. You can reach us
at tech_comm@waters.com.
Contacting Waters
®
Contact Waters with enhancement requests or technical questions regarding
the use, transportation, removal, or disposal of any Waters product. You can
reach us via the Internet, telephone, or conventional mail.
Waters contact information
Contacting medium Information

Internet The Waters Web site includes contact
information for Waters locations worldwide.
Visit www.waters.com.
Telephone and fax From the USA or Canada, phone 800
252-HPLC, or fax 508 872 1990.
For other locations worldwide, phone and fax
numbers appear in the Waters Web site.
Conventional mail Waters Corporation
34 Maple Street
Milford, MA 01757
USA
Safety considerations
Some reagents and samples used with Waters instruments and devices can
pose chemical, biological, and radiological hazards. You must know the
potentially hazardous effects of all substances you work with. Always follow
iii
Good Laboratory Practice, and consult your organization’s safety
representative for guidance.
Considerations specific to the Waters Quattro micro API

Solvent leakage hazard
The source exhaust system is designed to be robust and leak-tight. Waters
recommends you perform a hazard analysis, assuming a maximum leak into
the laboratory atmosphere of 10% LC eluate.
Warning:
• To confirm the integrity of the source exhaust system, renew
the source O-rings at intervals not exceeding one year.
• To avoid chemical degradation of the source O-rings, which can
withstand exposure only to certain solvents, see Appendix B to
determine whether any solvents you use that are not listed are
chemically compatible with the composition of the O-rings.
Flammable solvents hazard
Warning: To prevent the ignition of accumulated solvent vapors inside

the source, maintain a continuous flow of nitrogen through the source
whenever significant amounts of flammable solvents are used during
the instrument’s operation.
Never let the nitrogen supply pressure fall below 690 kPa (6.9 bar, 100 psi)
during analyses that require flammable solvents. Connect to the LC output
with a gas-fail connector to stop the LC solvent if the nitrogen supply fails.
iv
High temperature hazard
Warning: To avoid burn injuries, avoid touching the source enclosure

with your hand when operating or servicing the instrument.
Mass spectrometer high temperature hazard
Source enclosure
assembly
v
High voltage hazard
Warning:
• To avoid electric shock, do not remove the mass spectrometer’s
protective panels. The components they cover are not
user-serviceable.
• To avoid nonlethal electric shock when the instrument is in Operate
mode, avoid touching the areas marked with the high voltage
warning symbol. To touch those areas, first put the instrument in
Standby mode.
Mass spectrometer in ESI ionization mode
High voltage
connectors
Ion block and cone

assembly
ESI probe tip
vi
Hazards associated with removing an instrument from service
Warning: To avoid personal contamination with

biohazardous or toxic materials, wear chemical-resistant
gloves during all phases of instrument decontamination.
Warning: To avoid puncture injuries, handle syringes, fused silica lines,

and borosilicate tips with care.
When you remove the instrument from use to repair or dispose of it, you must
decontaminate all of its vacuum areas. These are the areas in which you can
expect to encounter the highest levels of contamination:
• Source interior
• Waste tubing
• Exhaust system
• Rotary pump oil (where applicable)
The need to decontaminate other vacuum areas of the instrument depends on
the kinds of samples the instrument analyzed and their levels of
concentration. Do not dispose of the instrument or return it to Waters for
repair until the authority responsible for approving its removal from the
premises specifies the extent of decontamination required and the level of
residual contamination permissible. That authority must also prescribe the
method of decontamination to be used and the appropriate protection for
personnel undertaking the decontamination process.
You must handle items such as syringes, fused silica lines, and borosilicate
tips used to carry sample into the source area in accordance with laboratory
procedures for contaminated vessels and sharps. To avoid contamination by
carcinogenic, toxic, or biohazardous substances, you must wear
chemical-resistant gloves when handling or disposing of used oil.
Safety advisories
Consult Appendix A for a comprehensive list of warning and caution
advisories.
vii
Operating this instrument
When operating this instrument, follow standard quality-control (QC)
procedures and the guidelines presented in this section.
Applicable symbols
Symbol Definition
Manufacturer
Authorized representative of the European

Community
Confirms that a manufactured product complies
with all applicable European Community
directives
Australia C-Tick EMC Compliant
Confirms that a manufactured product complies

with all applicable United States and Canadian
safety requirements
Consult instructions for use
For in vitro diagnostic use
Audience and purpose

This guide is intended for personnel who install, operate, and service the
TM
Waters Quattro micro API Mass Spectrometer.
viii
Intended use of the Waters Quattro micro API
The Waters Quattro micro API is CE-marked according to the
European Union In Vitro Diagnostic Device Directive 98/79/EC.
Waters designed the Quattro micro API for use as a research tool to deliver
authenticated exact-mass measurement on all sample types.
The Waters Quattro micro API can be used for general in vitro diagnostic
applications. However, only professionally trained and qualified laboratory
personnel can use the instrument for those purposes.
Calibrating
To calibrate LC systems, follow acceptable calibration methods using at least
five standards to generate a standard curve. The concentration range for
standards must include the entire range of QC samples, typical specimens,
and atypical specimens.
When calibrating mass spectrometers, consult the calibration section of the
operator’s guide for the instrument you are calibrating. In cases where an
overview and maintenance guide, not operator’s guide, accompanies the
instrument, consult the instrument’s online Help system for calibration
instructions.
Quality-control
Routinely run three QC samples that represent subnormal, normal, and
above-normal levels of a compound. Ensure that QC sample results fall within
an acceptable range, and evaluate precision from day to day and run to run.
Data collected when QC samples are out of range might not be valid. Do not
report these data until you are certain that the instrument performs
satisfactorily.
ix
When analyzing samples from a complex matrix such as soil, tissue,
serum/plasma, whole blood, and other sources, note that the matrix
components can adversely affect LC/MS results, enhancing or suppressing
ionization. To minimize these matrix effects, Waters recommends you adopt
the following measures:
• Prior to the instrumental analysis, use appropriate sample
pretreatment such as protein precipitation, liquid/liquid extraction
(LLE), or solid phase extraction (SPE) to remove matrix interferences.
• Whenever possible, verify method accuracy and precision using
matrix-matched calibrators and QC samples.
• Use one or more internal standard compounds, preferably isotopically
labeled analytes.
ISM classification
ISM Classification: ISM Group 1 Class A

This classification has been assigned in accordance with CISPR 11 Industrial
Scientific and Medical (ISM) instruments requirements. Group 1 products
apply to intentionally generated and/or used conductively coupled
radio-frequency energy that is necessary for the internal functioning of the
equipment. Class A products are suitable for use in commercial, (that is,
nonresidential) locations and can be directly connected to a low voltage,
power-supply network.
x
EC authorized representative
Waters Corporation (Micromass UK Ltd.)

Floats Road
Wythenshawe
Manchester M23 9LZ
United Kingdom
Telephone: +44-161-946-2400
Fax: +44-161-946-2480
Contact: Quality manager
xi
xii
Table of Contents
Copyright notice ................................................................................................... ii
Trademarks ............................................................................................................ ii
Customer comments ............................................................................................ iii
Contacting Waters ............................................................................................... iii
Safety considerations .......................................................................................... iii

Considerations specific to the Waters Quattro micro API................................ iv
Safety advisories ............................................................................................... vii
Operating this instrument .............................................................................. viii

Applicable symbols .......................................................................................... viii
Audience and purpose...................................................................................... viii
Intended use of the Waters Quattro micro API ................................................ ix
Calibrating .......................................................................................................... ix
Quality-control .................................................................................................... ix
ISM classification .................................................................................................. x

ISM Classification: ISM Group 1 Class A .......................................................... x
EC authorized representative ........................................................................... xi
1 Instrument Description ........................................................................ 1-1

Overview ............................................................................................................. 1-2
Sample inlet ........................................................................................................ 1-3
Vacuum system .................................................................................................. 1-3
Data system ........................................................................................................ 1-3
MassLynx software ........................................................................................... 1-4
Theory and principles of operation .............................................................. 1-4

ElectroSpray ionization (ESI) ......................................................................... 1-4
Atmospheric pressure chemical ionization (APcI) ......................................... 1-5
MS operating modes ........................................................................................ 1-5
Table of Contents xiii

MS/MS operating modes.................................................................................. 1-6
Front panel controls, indicators and connections .................................. 1-12

Cone gas, desolvation gas and nebulizer gas ............................................... 1-12
Electrical connections .................................................................................... 1-13
CID valve........................................................................................................ 1-13
Divert/injection valve..................................................................................... 1-14
Status display ................................................................................................ 1-15
Rear panel connections ................................................................................. 1-16

Analog channels ............................................................................................. 1-16
Contact closure............................................................................................... 1-17
Mux interface ................................................................................................. 1-17
Events............................................................................................................. 1-17
PC link............................................................................................................ 1-17
2 Routine Procedures ............................................................................... 2-1

Starting the Quattro micro API ..................................................................... 2-2
Installing the ESI (ElectroSpray) probe ...................................................... 2-5
Setting up the syringe pump .......................................................................... 2-6
Setting up the Quattro micro API ................................................................. 2-7

Preparing for ElectroSpray operation............................................................. 2-7
Obtaining an ion beam in ElectroSpray (ESI) mode.................................... 2-12
Preparing for APCI operation when in ESI mode........................................ 2-13
Obtaining an Ion beam and tuning in APCI mode ...................................... 2-14
3 Tuning ....................................................................................................... 3-1

Overview ............................................................................................................. 3-2
The tune page .................................................................................................... 3-2
Printing tune information .............................................................................. 3-2
Experimental record ........................................................................................ 3-3
Saving and restoring tune parameter settings .......................................... 3-4
Modifying the peak display ............................................................................ 3-5
xiv Table of Contents

Changing the display ....................................................................................... 3-8
Customize plot appearance ............................................................................. 3-9
Trace ............................................................................................................... 3-10
Intensity ......................................................................................................... 3-10
Grid................................................................................................................. 3-10
AutoTune ........................................................................................................... 3-10
Ion mode ............................................................................................................ 3-13
Scope parameters ............................................................................................ 3-13
Gas controls ...................................................................................................... 3-14

Ramp controls .................................................................................................. 3-15
Resetting the zero level ................................................................................. 3-17
Controlling readbacks ................................................................................... 3-17
Changing tune parameter settings ............................................................. 3-18

Source voltages ................................................................................................ 3-18
4 Data Acquisition ..................................................................................... 4-1

Starting an acquisition .................................................................................... 4-2
Starting an acquisition from the tune page ................................................... 4-2
Multiple samples.............................................................................................. 4-4
Automated quantification of sample list ........................................................ 4-6
Monitoring an acquisition .............................................................................. 4-8

The acquisition status (Scan Report) window ................................................ 4-8
Chromatogram real-time update .................................................................... 4-9
Spectrum real-time update ............................................................................. 4-9
Instrument data thresholds .......................................................................... 4-10

MaxEnt ........................................................................................................... 4-11
Profile data..................................................................................................... 4-11
Centroid data ................................................................................................. 4-12
SIR data.......................................................................................................... 4-12
Ion counting threshold................................................................................... 4-12
Profile data - spike removal .......................................................................... 4-14
Table of Contents xv
Analog data .................................................................................................... 4-15
System manager .............................................................................................. 4-16
Stopping an acquisition ................................................................................ 4-16

The function list editor .................................................................................. 4-16
Introduction.................................................................................................... 4-16
The Function List Editor toolbar .................................................................. 4-19
Adding a new function ................................................................................... 4-20
Modifying an existing function ..................................................................... 4-20
Copying an existing function......................................................................... 4-21
Removing a function ...................................................................................... 4-21
Changing the order of functions.................................................................... 4-21
Setting a solvent delay .................................................................................. 4-21
Analog channels ............................................................................................. 4-22
Saving and restoring a function list ............................................................. 4-23
Setting up a full scan function ...................................................................... 4-24
Setting up a SIR function .............................................................................. 4-28
Setting up MS/MS scanning functions ......................................................... 4-30
Setting up a MRM function ........................................................................... 4-32
Setting up a survey function ......................................................................... 4-33
5 Mass Calibration .................................................................................... 5-1

Introduction ....................................................................................................... 5-2
Overview ............................................................................................................. 5-2

Calibration types.............................................................................................. 5-4
The calibration process.................................................................................... 5-4
ElectroSpray ...................................................................................................... 5-5

Introduction...................................................................................................... 5-5
Preparing for calibration ................................................................................. 5-5
Calibration options .......................................................................................... 5-8
Selecting parameters ....................................................................................... 5-9
Performing a calibration................................................................................ 5-13
Checking the calibration ............................................................................... 5-18
ElectroSpray calibration with PEG .............................................................. 5-27
xvi Table of Contents

Atmospheric pressure chemical ionization (APcI) ................................. 5-29
Introduction.................................................................................................... 5-29
Preparing for calibration ............................................................................... 5-30
Calibration options ........................................................................................ 5-31
Selecting calibration parameters .................................................................. 5-31
Performing a calibration................................................................................ 5-32
Calibration failure ......................................................................................... 5-42
Incorrect calibration ...................................................................................... 5-44
Manual editing of peak matching ................................................................. 5-45
Saving the calibration.................................................................................... 5-45
Manual verification........................................................................................ 5-46
6 Maintenance Procedures ..................................................................... 6-1

Maintenance schedule ..................................................................................... 6-2
Safety and handling ......................................................................................... 6-3
Proper operating procedures ........................................................................... 6-3
Maintenance equipment .................................................................................. 6-3
Routine maintenance ....................................................................................... 6-4

Gas-ballasting the rotary pump ...................................................................... 6-4
Checking the rotary pump oil ......................................................................... 6-5
Changing the rotary pump oil ......................................................................... 6-6
Cleaning the source assembly ......................................................................... 6-7
Cleaning and replacing the corona discharge needle................................... 6-19
Cleaning the APCI probe tip ......................................................................... 6-22
Replacing the source enclosure and probe adjustment flange O-rings....... 6-22
Emptying the nitrogen exhaust waste bottle ............................................... 6-26
Replacing parts ............................................................................................... 6-28

Replacing the ion block cartridge heater...................................................... 6-28
Replacing the ESI probe stainless steel capillary ........................................ 6-30
Replacing the ESI probe tip .......................................................................... 6-32
Replacing the APcI probe heater .................................................................. 6-33
Replacing the APcI fused silica capillary and filter pad.............................. 6-33
Table of Contents xvii

7 Troubleshooting ..................................................................................... 7-1
Spare parts ......................................................................................................... 7-2
Safety and handling ......................................................................................... 7-2

System troubleshooting ................................................................................... 7-3
Component hardware troubleshooting ....................................................... 7-4

No peaks on the tune page (no ion beam)....................................................... 7-4
Unsteady or low intensity peaks (ion beam) .................................................. 7-5
Unusually high LC back pressure .................................................................. 7-7
Unusually low LC back pressure .................................................................... 7-7
Insufficient vacuum ......................................................................................... 7-8
Leaking nitrogen.............................................................................................. 7-9
Vacuum oil accumulated in the exhaust tubing............................................. 7-9
Source heater and desolvation heater not heating ........................................ 7-9
APcI heater not heating ................................................................................ 7-10
Roughing pump fuse fails .............................................................................. 7-10
Ion mode fault ................................................................................................ 7-11
Failure to recognize one particular probe type ............................................ 7-11
Ripple.............................................................................................................. 7-11
Loss of communication with instrument ...................................................... 7-12
IEEE communication errors.......................................................................... 7-13
High noise levels in MRM analyses ............................................................. 7-13

Chemical noise ............................................................................................... 7-14
Electronic noise ............................................................................................... 7-14
Contacting Waters .......................................................................................... 7-15
8 Reference Information ......................................................................... 8-1

Overview ............................................................................................................. 8-2
Editing a reference file .................................................................................... 8-2
Positive ion ......................................................................................................... 8-3

Horse heart myoglobin .................................................................................... 8-4
Polyethylene glycol .......................................................................................... 8-4
xviii Table of Contents

Sodium iodide and caesium iodide mixture.................................................... 8-5
Sodium iodide and rubidium iodide ................................................................ 8-5
Negative ion ....................................................................................................... 8-6

Sodium iodide solution for negative ion ElectroSpray................................... 8-6
A Safety Advisories .................................................................................. A-1

Warning symbols ............................................................................................... A-2
Task-specific hazard warnings........................................................................ A-2
Specific warnings ............................................................................................. A-3
Caution symbol .................................................................................................. A-5
Warnings that apply to all Waters instruments ......................................... A-6
Electrical and handling symbols ................................................................. A-11

Electrical symbols .......................................................................................... A-11
Handling symbols .......................................................................................... A-12
B Materials of Construction and Compatible Solvents ................... B-1

Preventing contamination ............................................................................. B-2
Items exposed to solvent ................................................................................ B-2

Solvents used to prepare mobile phases .................................................... B-3
C Environmental Specifications ........................................................... C-1

Access .................................................................................................................. C-2
Location .............................................................................................................. C-2

Quattro micro API ........................................................................................... C-2
Vacuum pump .................................................................................................. C-2
Data system...................................................................................................... C-3
LC system......................................................................................................... C-3
Dimensions and clearances ........................................................................... C-4

Height ............................................................................................................... C-6
Length .............................................................................................................. C-6
Width ................................................................................................................ C-6
Table of Contents xix

Weights ............................................................................................................... C-6
Lifting and carrying......................................................................................... C-6
Operating temperature .................................................................................. C-7
Operating humidity ......................................................................................... C-7
Shipping and storage temperature .............................................................. C-7
D Electrical Specifications .................................................................... D-1

Installation ........................................................................................................ D-2
Line frequency.................................................................................................. D-3
Fuse rating ....................................................................................................... D-4
Electrical safety ................................................................................................ D-4
E Performance Specifications ............................................................... E-1

ElectroSpray positive ion ............................................................................... E-1
ElectroSpray negative ion .............................................................................. E-1
APcI positive ion ............................................................................................... E-1
Index ..................................................................................................... Index-1
xx Table of Contents
1 Instrument Description
Contents
Topic Page
Overview 1-2
Sample inlet 1-3
Vacuum system 1-3
Data system 1-3
MassLynx software 1-4
Theory and principles of operation 1-4
Front panel controls, indicators and connections 1-12
Rear panel connections 1-16
1-1
Overview
The Waters Quattro micro API
Removable
Center Panel
TM
The Quattro micro API is a high performance triple quadrupole mass
spectrometer designed for routine LC/MS/MS operation.
The instrument may be coupled to the following liquid introduction systems:
• HPLC system, to provide molecular weight information from an LC run
or to perform target analysis and quantification.
• Syringe pump, for analysis of precious, low-concentration compounds.
Sample ionization takes place in the source at atmospheric pressure. These
ions are sampled through a series of orifices into the first quadrupole where
they are filtered according to their mass to charge ratio (m/z). The mass
separated ions then pass into the hexapole collision cell where they either
undergo collision induced decomposition (CID) or pass unhindered to the
second quadrupole. The fragment ions are then mass analyzed by the second
quadrupole.
The transmitted ions are finally detected by a conversion dynode, phosphor
and photomultiplier detection system. The output signal is amplified,
digitized and presented to the data system.
1-2 Instrument Description

Sample inlet
An HPLC system or an infusion pump delivers sample to either an
ElectroSpray Ionization (ESI) probe or Atmospheric Pressure Chemical
Ionization (APcI) probe.
The ionization mode can be changed by changing probes. Recognition pins on
the probes identify the ionization method to the system.
Vacuum system
An external rotary pump and an internal split-flow turbomolecular pump
combine to create a vacuum. The turbomolecular pump evacuates the
analyzer and ion transfer region.
The system monitors turbomolecular pump speed and continuously measures
the vacuum with a built-in Pirani gauge. In the event of leaks, electrical
failure, or vacuum pump failure a loss of vacuum will occur. The Pirani gauge
also acts as a switch, discontinuing instrument operation if it senses a vacuum
loss.
An easy-access vacuum isolation valve enables routine source maintenance to
be performed without breaking vacuum.
Data system
The data system collects information from the mass analyzer. The data
system consists of:
• An embedded PC
• An external workstation
• The MassLynx™ software
The workstation-based data system, incorporating MassLynx software,
controls the mass spectrometer and, if applicable, the HPLC system,
autosampler, divert valve or injector valve. The workstation uses the
Windows® graphical environment with color graphics, and provides for full
user interaction with either the keyboard or mouse. MassLynx provides full
control of the system including setting up and running selected HPLC
systems, tuning, acquiring data, and data processing. MassLynx instrument
Sample inlet 1-3

control uses an embedded PC to process all data. A network link enables
communication between the workstation and the embedded PC.
The data system can sample analog inputs and thus store data from
conventional LC detectors like UV or ELSD simultaneously with acquired
mass spectral data. It can also acquire UV photo diode array detector data for
selected systems such as the Waters 996 PDA. Comprehensive information
detailing the operation of MassLynx is in the MassLynx User’s Guide.
MassLynx software
MassLynx software, a Windows-based application, enables the following
operations:
• Configuring the Quattro micro API.
• Creating inlet and MS methods that define operating parameters for a
run.
• Tuning and calibrating the Quattro micro API.
• Running samples.
• Monitoring the run.
• Acquiring data.
Refer to the MassLynx User’s Guide and Help for more information on
installing and using MassLynx software.
Theory and principles of operation
ElectroSpray ionization (ESI)

In ElectroSpray ionization (ESI), a strong electrical charge is applied to the
eluent as it emerges from a nebulizer, producing an aerosol of charged
droplets. Solvent evaporation reduces the size of the droplets until a sufficient
charge density makes the ejection of sample ions from the surface of the
droplets possible (ion evaporation). Characteristically, ions are singly or
multiply charged, and the mass analyzer sorts them by mass-to-charge (m/z)
ratio. High molecular weight compounds are typically measured as ions with
multiple charges. Eluent flows up to 1 mL/min can be accommodated, though
it is often preferable with ElectroSpray ionization to split the flow so that 100
to 200 µL/min of eluent enters the mass spectrometer source.

Atmospheric pressure chemical ionization (APcI)
APcI generally produces protonated or deprotonated molecular ions from the
sample via a proton transfer (positive ions) or proton abstraction (negative
ions) mechanism. The sample is vaporized in a heated nebulizer before flowing
into a plasma consisting of solvent ions formed within the atmospheric source
by a corona discharge. Proton transfer then takes place between the solvent
ions and the sample. Eluent flows up to 2 mL/min can be accommodated
without splitting the flow.
MS operating modes
Ion optics
Source MS1 Collision Cell MS2 Detector
MS operating modes
Operating mode MS1 Collision cell MS2

MS1 Resolving Radio Frequency (RF) only (pass all
masses)
MS2 Radio Frequency (RF) only (pass all Resolving
masses)
The MS1 mode, in which MS1 is used as the mass filter, is the most common
and most sensitive method of performing MS analysis. This is directly
analogous to using a single quadrupole mass spectrometer.
The MS2 mode of operation is used, with collision gas present, when switching
rapidly between MS and MS/MS operation. It also provides a useful tool for
instrument tuning and calibration prior to MS/MS analysis, and for fault
diagnosis.

MS/MS operating modes
General
The four common MS/MS scan functions are summarized in the table below.
MS/MS operating modes
Operating mode MS1 Collision cell MS2

Daughter Static Scanning
(Product) Ion (parent mass
Spectrum selection)
Parent Scanning Static
(Precursor) Ion (daughter mass
Spectrum selection)
RF only (pass all
Multiple Static masses) Static
Reaction (parent mass (daughter mass
Monitoring selection) selection)
Constant Neutral Scanning Scanning
Loss Spectrum (synchronized (synchronized
with MS2) with MS1)
The Daughter (product) ion spectrum
Daughter (product) ion mode
MS1 Collision Cell MS2

Static at parent mass RF only Scanning
(pass all masses)
This is the most commonly used MS/MS scan mode.

Typical applications are:
• Structural elucidation (for example, peptide sequencing).
• Method development for MRM screening studies:
– Identification of daughter ions for use in MRM transitions.
– Optimization of CID tuning conditions to maximize the yield of a
specific daughter ion to be used in MRM analysis.
Example:
Daughters of the specific parent at m/z 609 from reserpine in ElectroSpray
positive ion mode, see the figure “Daughters of the specific parent at m/z 609
from reserpine in ElectroSpray positive ion mode” on page 1-7.
Daughters of the specific parent at m/z 609 from reserpine in ElectroSpray

positive ion mode

The Parent (precursor) ion spectrum
Parent (precursor) ion mode

Scanning RF only Static at daughter mass
(pass all masses)
Typical application:
• Structural elucidation.
– Complementary or confirmatory information (for daughter scan
data).

Example:
Parents of the specific daughter ion at m/z 195 from reserpine in ElectroSpray
positive ion mode, see figure below.
Parents of the specific daughter ion at m/z 195 from reserpine in

ElectroSpray positive ion mode

MRM: multiple reaction monitoring
MRM mode

Static at parent mass RF only Static at daughter mass
(pass all masses)
This mode is the MS/MS equivalent of SIR (Selected Ion Recording). As both
MS1 and MS2 are static, this allows greater dwell time on the ions of interest
and therefore better sensitivity compared to scanning MS/MS.
• Rapid screening of dirty samples for known analytes:
– Drug metabolite and pharmacokinetic studies.
– Environmental, for example pesticide and herbicide analysis.
– Forensic or toxicology, for example screening for target drugs in
sport.
Example:
Monitor the transition (specific fragmentation reaction) m/z 609 to 195 for
reserpine in ElectroSpray positive ion LC/MS/MS mode.
MRM does not produce a spectrum as only one transition is monitored. As in
SIR, a chromatogram is produced.

The m/z 609 to 195 transition for reserpine in ElectroSpray positive ion
LC/MS/MS mode
Time Time
LC/MRM LC/MS
High specificity Low specificity
Good signal/noise Poor signal/noise
The constant neutral loss spectrum
Collision Cell
RF only
MS1 (pass all masses)
MS2
scanning scanning
The loss of a specific neutral fragment or functional group from an unspecified

parent or parents.
• Screening mixtures for a specific class of compound that is characterized
by a common fragmentation pathway.
The scans of MS1 and MS2 are synchronized. When MS1 transmits a specific
parent ion, MS2 “looks” to see if that parent loses a fragment of a certain
mass. If it does, it registers at the detector.
The result:
The spectrum shows the masses of all parents that actually lost a fragment of
a certain mass.

Front panel controls, indicators and connections
Front panel
Desolvation Gas Fused Silica Guide
Nebulizer Gas
CID Valve Injector/Divert Valve
Syringe Pump
Fused Silica Guide
Reset Switch
Cone gas, desolvation gas and nebulizer gas

The PTFE gas lines for the desolvation gas and nebulizer gas are connected to
the front of the instrument using push-in Legris fittings. The connection for
the cone gas is within the source and uses PTFE tubing.
Mass flow controllers

Electronic mass flow controllers, whose settings you specify in the Tune
window, regulate the cone gas and desolvation gas over the ranges 0 to
500 L/h and 0 to 1200 L/h, respectively.
In the event that the desolvation gas flow decreases to less than 4% of its full
scale range, the instrument generates a signal that can be used to stop solvent
flowing into the source by connecting it to the Stop Flow of the HPLC system.

Electrical connections
The electrical connection for the APcI probe or the ESI heater is via the
ESI / APcI multi-way connector. This is removed from the front panel by
pulling on the metal sleeve of the plug. Both the ElectroSpray and APcI
heaters use this connector.
The high voltage connection for the ESI probe is via the front panel ESI
connection.
The high voltage connection for the corona discharge pin is internal to the
source.
Caution: Ensure that the instrument is in Standby when fitting the

corona discharge pin.
CID valve
The CID Gas valve is a fifteen-turn valve. The collision gas flow increases as
the valve is turned counterclockwise.
Caution: To prevent damage to the CID Gas valve, take care not to
over-tighten when turning the supply off.

Divert/injection valve
Divert/injection valve
HPLC Probe
Pump
Sample Loop
Sample Waste
Syringe
®
The divert/injection valve is an electrically driven Rheodyne injector that
may be used in several ways depending on the plumbing arrangement:
• As an injection valve, with the needle port and sample loop fitted.
• As a divert valve, to switch the flow of solvent during a LC run.
• As a switching valve, for example to switch between a LC system and a
syringe pump containing calibrant.
Control of the valve is primarily from the data system. The two switches
marked Load and Inject enable the user to override control of the valve when
making loop injections at the instrument.
For details of the use of the valve as a divert valve see page 4-21.

Status display
Status display
Vacuum
Operate
The status of the instrument is indicated as follows:
Vacuum LED
State Vacuum LED

Pumping Flashing green
Pumped, below trip level Steady green
Pumped, above trip level Steady amber
Pump fault Flashing red
Operate LED
State Operate LED

Standby No indication
Operate, above trip level Steady amber
Operate, below trip level Steady green
RF error Flashing red

Rear panel connections
Rear panel connections
ANALOGUE CHANNELS CONTACT CLOSURE EVENTS
CH1 CH2 EVENT 1 EVENT 2 CE INT GF !

OUT CAUTION!
+
-
REFER TO
CH3 CH4 EVENT 1 EVENT 2 FC MANUAL BEFORE
CONNECTING TO
IN THESE PORTS
+
-
CONSULTER
MUX INTERFACE LE MANUEL
AVANT DE
BRANCHER
LES
PC LINK
CONNECTTEURS
Analog channels
Four analog channel inputs are available, for acquiring simultaneous data
such as a UV detector output.
The input differential voltage must not exceed one volt. Analogue data is
20
processed by a 12-bit ADC with a gain ranging up to 2×10 counts.
If the input cable is only a two-wire assembly, then the negative pole of each
channel may need to be grounded.

Contact closure
Two types of contact closure are available:
• In. Two inputs, Event 1 and Event 2, are provided, allowing external
devices to start acquisition. The Event In signal can be TTL or contact
closure, 5 V maximum voltage.
• Out. Two outputs, Event 1 and Event 2, are provided whereby the mass
spectrometer is able to trigger an external event.
Mux interface
This 9-way D-type connector enables interfacing to the MUX control unit.
Events
CE int (capillary electrophoresis interlock)
This connector enables interfacing with a capillary electrophoresis power
supply so that the instrument is safely interlocked against high voltages.
GF (gas fail)
If the desolvation gas flow decreases to less than 4% of its full-scale range, the
instrument generates a signal that can be used to stop solvent flowing into the
source by connecting it to the Stop Flow of the HPLC system.
FC (FractionLynx control)
A 100 mV analog output signal is provided to allow a trigger signal for an
external fraction collection device. The optional FractionLynx software must
be purchased for this.
PC link
This RJ45 connector links the instrument to the data system using the
network cable supplied.
Rear panel connections 1-17

2 Routine Procedures
Contents
Topic Page
Starting the Quattro micro API 2-2
Installing the ESI (ElectroSpray) probe 2-5
Setting up the syringe pump 2-6
Setting up the Quattro micro API 2-7
2-1
Starting the Quattro micro API
To start the Quattro micro API proceed as follows:

1. Switch on the switch located under the left hand side of the front panel.
On/off switch
On/Off
Switch
2. Allow 3 minutes for the embedded PC to initialise. An audible alert is

given when the PC is ready.
3. Start the MassLynx software. The default page appears, and the word
Ready appears in the status bar at the bottom.
2-2 Routine Procedures

MassLynx default page
4. Click (on the default page) to invoke the tune page.

5. Select Pump from the tune page Options menu.
6. Click the Diagnostics tab.
7. Monitor Turbo Speed. This parameter should reach 98 to 100% within
approximately 5 minutes of Pump being selected.
8. Ensure that the instrument has pumped sufficiently such that the
Vacuum LED on the front panel is steady green (see page 1-15). The
mass spectrometer is sufficiently evacuated to enable operation in
20 minutes.
Starting the Quattro micro API 2-3

Tune page

Installing the ESI (ElectroSpray) probe
ESI probe
Probe
High Voltage Lead
Desolvation Lead
Gas
Knurled
Thumbscrews
(2)
Probe
Nebulizer
Gas Flange
CID
Valve Probe Adjuster
Plate
Probe
Adjuster
To install the ESI probe:

1. Ensure that the isolation valve lever is fully to the left, indicating the
valve is open.
2. Insert the probe adjustment flange electrical cable in the lower (and
larger) of the two electrical ports on the front panel.
3. Connect the PTFE tubing from the probe adjustment flange to the
desolvation gas port on the front panel.
4. Remove the protective sleeve, if fitted, from the ElectroSpray probe tip.
5. Slide the ElectroSpray probe into the hole in the probe adjustment
flange until the probe body rests on the probe adjustment flange. Ensure
the probe identification contacts touch the screws on the probe
adjustment flange.
6. Secure the probe with the two thumbscrews.
Installing the ESI (ElectroSpray) probe 2-5

7. Connect the 4 mm PTFE tubing from the probe to the port labelled Neb
(nebulizer gas).
8. Connect the electrical lead from the probe to the capillary connector on
the front panel.
Setting up the syringe pump
Syringe pump
Syringe
Needle Port
Capillary
Syringe Stop
Adjuster
To set up the syringe pump:

1. Clip the ground cable (with plug-in clip), located at the front panel,
lower right, onto the syringe needle.
2. Mount the syringe onto the pump, and set the syringe stop
appropriately.
Caution: Waters has incorporated into the syringe pump design a

positive syringe stop to prevent certain syringe types from
breaking. Nevertheless, as added protection against syringe
breakage, setting the syringe stop adjuster is recommended. This
prevents the syringe plunger from travelling its full stroke inside
the syringe barrel, thereby reducing the likelihood of breakage.
3. Screw the Rheodyne 9013 needle port fitting into the PEEK™ union,
and tighten it so that it does not leak.

4. Feed the capillary (ESI probe installation kit) from the top of the
molding to the syringe area. Connect the capillary to the PEEK union,
using an Upchurch® Scientific nut, ferrule, and PTFE tubing.
5. Make a square, even cut on both ends of the capillary before installing,
using a ceramic silica cutter. Examine new cuts for squareness using an
eye glass. When cutting the capillary, allow enough length to form loops
at angles and corners. Never kink the capillary or stretch it tightly from
one point to another.
Warning: Clip the ground cable (with plug-in clip), located at the
front panel, lower right, onto the syringe needle.
6. Connect the other end of the capillary to the inlet on the ESI probe with
an Upchurch Scientific nut, ferrule, and PTFE tubing.
7. Click (on the default page) to access the tune page.

8. Choose a suitable syringe type from the syringe selection editor by
selecting Options, then Syringe Type, from the tune page.
Setting up the Quattro micro API
Preparing for ElectroSpray operation

Warning: Be sure to ground the syringe needle with the ground cable
provided.
To prepare for ElectroSpray operation:

1. Connect one end of the fused silica capillary tubing to the syringe, and
connect the other end to the ESI probe.
2. Fill the syringe with a reference solution, and mount it on the syringe
pump.
3. Click on the default page to invoke the tune page. The example tune
page, shown in the figure “ESI tune page” on page 2-9, uses ions from a
solution of PPG1000, reserpine and PA ß Cyclodextrin.

Default page
4. Select Options from the menu bar, then Pump. The rotary pump starts
to evacuate the detector. In about 20 minutes, the instrument is
sufficiently evacuated to enable operation, and the Vacuum indicator on
the front panel shows green.
5. To view the actual values for instrument parameters select Readbacks
from the Options menu, then Always On.
6. Enter the suggested initial reference solution values from the table
titled “Tune page initial reference solution values” on page 2-9 in the
corresponding tune page fields. These settings are intended as starting
points only. Optimum values may vary between instruments.

ESI tune page
Tune page initial reference solution values
Mass Span Gain

175.1 5 8
609.2 5 20
1080.8 5 40
2034.6 5 50

7. Enter the recommended parameter values from the table titled “ES+
source page recommended parameter values” on page 2-10 in the
corresponding fields of the ES+ Source page.
Caution:
• Failure to flow desolvation gas during ESI operation can cause
heat damage to the source.
• To avoid contaminating source components, always specify a
desolvation gas flow rate that exceeds 100 L/h.
ES+ source page recommended parameter values
Parameter Suggested value

Capillary (kV) 3.0
Cone (V) 60
Extractor (V) 3
RF Lens (V) 0.2
Source Temperature (°C) 120
Desolvation Gas (L/h) 150
Cone Gas (L/h) 0
Tip: To avoid nitrogen leakage, fully tighten the probe locking ring.
8. Click the Analyser tab.
9. Enter the parameter values listed in the following table, dependent on
whether tuning for MS1 or MS2.
Analyser parameter values
Recommended value Recommended value

Parameter
MS1 MS2
LM Resolution 1 15
HM Resolution 1 15
Ion Energy 1 0.5
Entrance 50 2
Collision 0 0
Exit 50 2

Analyser parameter values (Continued)
Recommended value Recommended value

Parameter
MS1 MS2
LM Resolution 2 15 15
HM Resolution 2 15 15
Ion Energy 2 3 0.5
Multiplier 650 650
Analyser page
10. Suitable resolution can be obtained by adjusting LM Resolution and HM

Resolution.
11. Click to start the nitrogen flow.

Obtaining an ion beam in ElectroSpray (ESI) mode
To obtain an ion beam in ElectroSpray (ESI) mode:
1. Ensure that the ESI probe is installed as described on page 2-5.
2. Change the ionization mode to ESI, if necessary (the current tune page
tab indicates the ionization mode):
Select Ion Mode > Electrospray+ from the tune page menu.
3. Keep the tune page ES+ Source tab open for the remaining steps in this
section.
4. Set Source Temp to 120 °C.
5. Allow the source temperature to reach 120 °C.

7. From the Options menu, select the type of syringe to be used. For
example, select the Hamilton 250-µL gas-tight syringe from the startup
kit.
8. Click Press For Operate to switch on the instrument high voltages.
9. Set the syringe flow rate to 10 µL/min., and click on the tune page
menu bar.
10. On the tune page, set Desolvation Gas to 150 L/h.
11. Check for leaks at the probe and syringe fittings.
12. Monitor for mass peaks. The peaks should appear at approximately the
mass values entered on the ES+ Source tab.
13. Increase values in the Gain fields until mass peaks become clearly
visible.
Caution:
• To avoid contaminating source components, always specify a
• An optimum signal must be obtained before the instrument can
successfully be calibrated.

14. If the signal is relatively weak and noisy, enhance it by turning the
probe adjuster knob to adjust the orientation of the probe relative to the
sample cone orifice. The signal can also be enhanced by adjusting the
desolvation gas flow from the ES+ Source tab on the tune page
Caution: If the nitrogen supply to the instrument is turned off

overnight, be sure the API Gas parameter on the tune page is set
to Off before restarting nitrogen flow. Failure to do this may
damage the flow meter.
The source is now ready for ElectroSpray use. Refer to Chapter 3 for
further information.
Preparing for APCI operation when in ESI mode

To prepare for APCI operation when in ESI mode:
1. Switch the instrument into standby mode by clicking Press for Standby
on the lower right of the tune page.
2. Switch the instrument into standby mode by clicking Press for Standby
on the lower right of the tune page.
3. Disconnect the nebulizer and both electrical connections from the front
panel.
4. Remove the ESI probe by unscrewing the two thumb nuts on the probe.
Caution: To avoid damaging the source enclosure door, do not

apply any force to it while it is open.
5. Remove the middle moulding section, unfasten the source enclosure

door’s securing clips, and open the source enclosure door.
6. Close the source enclosure door, fasten the securing clips, and fit the
middle molding section.
7. With the corona discharge pin in place, proceed as follows:
a. Insert the APCI probe into the source and tighten the two
thumbscrews.
b. Connect the 6-mm nebulizer gas tube from the probe to the
instrument port marked Neb.

c. Remove the probe adjustment flange cable from the front panel, and
seat it in the rest hole provided just below its electrical socket.
d. Connect the APCI probe electrical lead to the Source/Probe
receptacle on the front panel.
e. Connect the LC pump tubing to the APCI probe.
f. Set the Source Temp to 130 °C.
g. Set APCI Probe Temp to 20 °C with zero liquid and nitrogen flow.
h. Switch the instrument to Operate.
The source is now ready for APCI operation.
Caution:
• Before restarting nitrogen flow following its interruption, the
API gas flow must be stopped from the tune page. Restarting
the nitrogen while the API gas is flowing can damage the flow
meter.
• Do not start the liquid flow until the gas flow and probe heater
are switched on with the probe inserted.
Obtaining an Ion beam and tuning in APCI mode

To obtain an ion beam:
1. Ensure that the corona discharge pin is in place, and the APCI probe is
installed as described on page 2-13.
2. Change the ionization mode to APCI, if necessary. Select Ion Mode from
the tune page menu. The current tune page tab indicates the ionization
mode.

3. Keep the tune page APCI+ Source tab open.
APCI tune page
4. Ensure that the desolvation gas tube is connected at the front panel.
5. Set Source Temp to 130 °C.
6. Set Corona to 2 µA and Sample Cone to 50 V.
7. Allow the source temperature to reach 130 °C.

9. Set Desolvation Gas to 250 L/h on the APCI+ source tune page.
10. Select one of the peak display boxes, and set Mass to 50 and Span to 90.
11. Click Press to Operate.

12. Set APCI Probe Temp to 500 °C for acetonitrile:water 1:1 flowing at
1 mL/min.
Lower temperatures are required for higher proportions of organic
mobile phase.
13. Allow the APCI probe temperature to reach 500 °C.
14. Start the LC pump flowing at 1.0 mL/min.
15. Adjust the spray approximately to midway between the corona pin and
the sample cone with the probe adjuster.
Refer to page 2-16 for more information on source tuning.
Warning:
• The source enclosure and parts of the probe adjustment flange
may reach high temperatures when in use.
• Switch off the liquid flow and allow the probe to cool to less
than 100 °C before removing it from the source.
Caution: Failure to flow desolvation gas during ESI operation can

cause heat damage to the source.
Performing a sample analysis

Typical parameters for general qualitative analysis of mixtures are shown in
the table titled “Typical parameters for general qualitative analysis of
mixtures” on page 2-17. Adjust the values of Corona, Cone and APCI Probe
Temp for optimal performance.

Specific tuning for maximum sensitivity
Caution: To avoid contaminating source components, always specify a

desolvation gas flow rate of greater than 100 L/h.
Typical parameters for general qualitative analysis of mixtures
Parameter Typical value

Corona (µA)* 2
Cone (V) 25 (Monitor the ions, slide the adjuster up or
down to optimize)
Extractor (V) 5
RF Lens (V) 0.2
Source Temp (°C) 130
APCI Probe Temp (°C)* 500
Desolvation Gas (L/h)* 300
Cone Gas (L/h)* 100
* See page 2-17 for specific tuning details.
For quantitative analysis, optimum APCI conditions should be obtained for

each analyte using standard solutions.
Tuning at high flow rates in APCI may be performed using a tee to introduce a
standard solution (typically 100 to 1000 pg/µL) at 10 µL/min into the mobile
phase stream.
Alternatively, repeat direct loop injections of a standard solution (typically 10
to 100 pg/µL) into the mobile phase stream may be used to optimize the APCI.
Probe position
Turn the probe flange adjuster to optimize the signal. Spray should be
approximately midway between the corona pin and the sample cone.
Corona current
Corona current can have a significant effect on sensitivity. The corona current
required depends upon the polarity of the compound and the polarity of the
analytical mobile phase. Optimization should be performed in the presence of
the analytical mobile phase.

For polar compounds analyzed in a polar mobile phase, the signal may be
improved by reducing the corona current below 2 µA.
For compounds of low polarity analyzed in a low polarity mobile phase, the
signal may be improved by increasing the corona current above 2 µA.
To find the optimum corona current value:

1. Set Corona Current to 2 µA.
2. Increase Corona Current value in 2 µA steps until the optimum value is
found. Allow the current to stabilize before taking a reading.
3. If the signal continuously decreases, return Corona Current to 2 µA,
then reduce the value in 0.5 µA steps until the optimum value is found.
Using Corona Current values greater than 0 µA will yield the best results for
most samples of this type.
Probe temperature
For maximum sensitivity, the APCI probe temperature must be optimized as
follows, ensuring that the analytical mobile phase is used during optimization:
Starting at 650 °C, reduce APCI Probe Temp in 50 °C decrements,
allowing time for the temperature to stabilize before taking a reading.
It is possible to set APCI Probe Temp too low for the mobile phase. This often
results in significant tailing of chromatographic peaks.
Desolvation gas
Caution: To avoid contaminating source components, always specify a

In most circumstances the desolvation gas flow has little effect on signal
intensity. However, in some situations, it can affect chemical background
noise levels. Adjusting desolvation gas can suppress chemical background
noise.
Cone gas
Set the cone gas flow to minimize formation of solvent adducts. The typical
value is about 50 L/h.

3 Tuning
Contents
Topic Page
Overview 3-2
The tune page 3-2
Printing tune information 3-2
Experimental record 3-3
Saving and restoring tune parameter settings 3-4
Modifying the peak display 3-5
Changing the display 3-8
AutoTune 3-10
Ion mode 3-13
Scope parameters 3-13
Gas controls 3-14
Ramp controls 3-15
Resetting the zero level 3-17
Controlling readbacks 3-17
Changing tune parameter settings 3-18
Source voltages 3-18
3-1
Overview
For the highest mass accuracy, the instrument should be tuned and calibrated
using a suitable reference compound before sample data are acquired.
• Consult the relevant section of this manual for information concerning
source tuning procedures in the chosen mode of operation.
• Adjust the tuning parameters in the Source and Analyser menus to
optimize peak shape and intensity at unit mass resolution.
• Care should be taken to optimize the value of the collision energy. Note
that, in Daughter and Parent modes, Collision and Exit are interactive
parameters.
The tune page
To invoke the tune page, press on the MassLynx screen MS panel.

Refer to the figure “Tune page” on page 3-3 for details of the tune page layout.
Printing tune information

To print a report, containing a copy of the tune peak information invoked on
the screen along with a record of each parameter setting, press , or select
Print from the tune page File menu.
This report is not configurable by the user.
3-2 Tuning
Tune page
Experimental record
Tuning parameters are stored with every data file as part of the experimental
record. The tuning parameters for a particular data file can be viewed or
printed from the data browser, see the MassLynx NT User’s Guide for more
information.
Experimental record 3-3

Saving and restoring tune parameter settings
Whole sets of instrument tuning parameters can be saved to disk as a named
file and then recalled at a future date.
A tune parameter file contains the latest settings for the source controls for all
supported ionization modes not just the ionization mode currently selected.
Tune parameter files also contain settings for the analyser, inlet set points
and peak display.
To save the current tune parameters with the existing file name:
1. Press , or choose Save from the tune page File menu.

2. Press Save.
To save the current tune parameters with a new file name:

1. Select Save As from the tune page File menu; the Save As dialog box is
opened.
Save As dialog box
2. Enter a new file name, or select an existing file from the displayed list.
3-4 Tuning
3. Press Save.
4. If the selected file already exists on disk, a warning is displayed. Press
Yes to overwrite the existing information or No to enter a different file
name.
To restore a saved set of parameters:
1. Press , or choose Open from the tune page File menu, the Open dialog
box is opened.
Open dialog box
2. Select the required tuning parameter file, either by typing its name or
by selecting from the list.
3. Press Open.
Modifying the peak display

The tune peak display is modified using either the tune peak controls, or the
mouse directly on the display.

To select peaks:
1. Press , or select Options, Peak Editor.

2. Choose the peaks to be displayed by checking the appropriate boxes.
3. For each active peak select the Mass, Span and Gain.
To change the function:

1. Select the function for the peak from the drop-down list.
For MS/MS functions, Set is enabled allowing the mass of the parent,
daughter, neutral loss or neutral gain ion to be entered.
To change the tune mass:
Either:
1. Click and drag the mouse within the bounds of the axis to draw a
“rubber band” around the region of interest.
2. Release the button.
This range is redisplayed to fill the window. The mass displayed in the
Mass box is the mass at the centre of the window.
This operation can be repeated as often as required.
3. Pressing once displays the previous magnification range and mass,

pressing it a second time returns to the default settings.
Or:
1. Enter a value in the Mass box for the required peak and press Return.
This becomes the default, so, if the range is altered with the mouse and
is pressed twice, Mass returns to this value.
Or:
1. Position the cursor at the top of the peak window, just below the line
showing the gain.
2. When appears, click the left mouse button and drag until the
required mass is displayed in the Mass box and at the top of the window.
3-6 Tuning
This becomes the default, so if the range is altered with the mouse and
is pressed twice Mass returns to this value.
Or:
1. Position the cursor at the top of the peak window, just below the line
showing the gain.
2. When appears, click the left mouse button and drag until the
required mass is displayed in the Mass box and at the top of the window.
is pressed twice Mass returns to this value.
To change the span of a peak:
Either:
1. Press the left mouse button at one end of the region of interest and,
without releasing the button, drag the mouse horizontally to the other
end.
As the mouse is dragged a “rubber band” stretches out to indicate the
selected range.
Do not go beyond the bounds of the axis.
2. Release the mouse button to re-display the selected range filling the
current window.
This operation can be repeated as often as required.
Pressing once displays the previous magnification range, pressing it

a second time returns to the default settings.
Or:
1. Enter a value in the Span box for the required peak and press Return.
is pressed twice Span returns to this value.

To change the gain of a peak:
Either:
1. Double-click on the line above the peak which shows the gain, to double
the gain applied to that peak.
2. Double-click below the peak to halve the gain.
Or:
1. Press the left mouse button at one end of the region of interest and,
without releasing the button, drag the mouse vertically to the other end.
As the mouse is dragged, a marquee indicates the selected range.
Do not go beyond the bounds of the axis.
2. Release the mouse button to re-display the selected range filling the
current window.
Or:
1. Enter a value in the Gain box for the required peak and press Return.
Changing the display
To change the display using the mouse:

• Click in the peak display area with the right mouse button to invoke the
Peak Display pop-up menu.
Peak display pop-up menu
The display area for each peak can be individually changed, e.g. the peak color
for peak 1 can be red, for peak 2 green, etc.
3-8 Tuning
Customize plot appearance
To change the color of the background and traces and to change the
number of traces displayed:
• Select Customise, Plot Appearance from the Peak Display pop-up menu,
see the figure “Peak display pop-up menu” on page 3-8.
• The Customise Plot Appearance dialog box is invoked, see figure below.
Customise Plot Appearance dialog box
To change the colors on the display:

• Press Newest Trace, Background, or Trace Fill, and select a new color
from the invoked dialog box.
To change the number of traces:
• Use to change the number, or enter a new value in the Visible Traces
box, within the range 2 to 20.
• If more than one trace is displayed, the older traces can be displayed in a
different shade to the new ones:
Changing the display 3-9

Drag the Colour Interpolation slider toward the full position. The color
of the old traces is shown in the Trace colour sample (new->old) field.
Trace
From the Peak Display pop-up menu, either:
Select the Trace, Outline option to display the peak outline only.
Or:
Select the Trace, Fill option to fill the trace with the trace fill colour.
Or:
Select the Trace, Min/Max option to show the minimum and maximum
data points only.
The option selected has a tick next to it.
Intensity
To select intensity:
1. From the Peak Display pop-up menu, select either Intensity,
Relative Intensity, or Intensity, Absolute Intensity as required.
2. Select Intensity, Normalise Data to display normalized data.
The options selected each have a tick next to them.
Grid
The Peak Display pop-up menu, options allow vertical and horizontal grid
lines to be independently displayed or hidden.
Selected options have ticks next to them. Selecting an option a second time
deselects the option.
AutoTune
MassLynx can automatically tune the mass spectrometer in both APcI and
ElectroSpray ionization modes. AutoTune ramps the settings for the tuning
parameters until they are optimized to give the best intensity, resolution and
peak shape.
3-10 Tuning
To run AutoTune:
1. Press on the tune page to turn on the API gas, and select Operate.
2. Choose AutoTune from the tune page Options menu, the AutoTune
dialog box is invoked.
AutoTune dialog box
3. Press Setup to define the AutoTune setup parameters, the AutoTune

Setup dialog box is invoked.
There are two levels of AutoTune:
• A Full AutoTune starts from a default set of tuning parameters.
• A Maintenance AutoTune starts from the current tuning parameters set
in the tune page and can be quicker than a full AutoTune.
A Maintenance AutoTune can only be performed if the instrument is
already reasonably well tuned. If the current tuning is too poor,
AutoTune gives an error and requests a Full AutoTune.
AutoTune 3-11
AutoTune Setup dialog box
The Tune Mass parameter sets the mass to be tuned on.
When satisfied with the AutoTune setup parameters:

1. Press OK to exit.
2. Press Start.
The AUTOTUNE STATUS bar is updated to show the progress of the
AutoTune.
The following steps are performed:
• Parameter initialization and instrument checks:
– Ensuring that essential status indicators read correctly.
– Checking that values are defined for all the user controllable
instrument parameters and that these are passed to the data
system.
– Checking that readbacks for these parameters are within specified
tolerances.
• Beam detection
• Focus lens tuning
3-12 Tuning
• Ion energy tuning
• High and low mass resolution tuning
– The final four of these steps represent the implementation of the
ESP/APcI AutoTune algorithm. This involves changing key
parameters, one at a time, to maximize the intensity of a reference
peak with respect to that parameter. At present, ESP/APcI
Autotuning is carried out with respect to a single user-specified
reference peak.
When AutoTune has finished, it displays a status window to say that
AutoTune has been successfully completed.
Press OK to return to the tune page.
The tuning parameters determined by AutoTune are saved to the
current tune parameter file.
Ion mode
Select the required ionization mode from the Tune Page Ion Mode menu. The
selected mode has a tick next to it.
Scope parameters
The Scope Setup dialog box is invoked by pressing , or selecting the Tune
Page Options, Scope Parameters menu.
Scope Setup dialog box
Scan Time(s) and Inter Scan Delay (s) control the speed with which the tune
peak display is updated.
Ion mode 3-13

Tuning is more responsive when these parameters are low in value.
To change the scope parameters:
1. Press , or choose Scope Parameters from the tune page Options

menu.
2. Make any required changes to the settings.
3. Press OK.
Gas controls
To turn a gas on or off:
Press (for nebulizer, desolvation and cone gas), or (for collision

gas), or choose the required gas from the tune page Gas menu.
If the gas was previously turned off it is now turned on. A tick mark
appears next to a gas if it is turned on.
3-14 Tuning
Ramp controls
To set up a cone voltage ramp:

1. Choose Cone Ramp Gradient from the tune page Ramps menu, the Cone
Ramp dialog box is invoked.
Cone Ramp dialog box
Two values of cone voltage are defined at two particular masses. These
values define a gradient for the cone voltage which is then extrapolated
to cover the full mass range.
2. Make any changes required and press OK to exit.
To initiate the cone voltage ramp:
1. Press , or choose Use Cone Ramp from the tune page Ramps menu.
A tick mark appears next to the menu item if the cone voltage ramp is
selected.
Ramp controls 3-15

To set up a collision energy ramp:
1. Choose Collision Energy Ramp Gradient from the tune page Ramps
menu, the Collision Ramp dialog box is invoked.
Collision Ramp dialog box
Two values of collision energy are defined at two particular masses.

These values define a gradient for the collision energy voltage which is
then extrapolated to cover the full mass range.
2. Make any changes required and press OK to exit.
To initiate the collision energy voltage ramp:
1. Press , or choose Use Collision Energy Ramp from the tune page
Ramps menu.
3-16 Tuning
Resetting the zero level
The zero level (or baseline) can be repositioned by pressing , or by choosing

Reinitialize from the tune page Options menu.
This command causes the instrument control system to measure the position
of the noise signal so that any baseline offset caused by the electronics or
instrumentation can be compensated for.
It is advisable to reset the zero level whenever the multiplier voltage is
changed.
Controlling readbacks
The Readbacks dialog box is invoked by selecting the Tune Page Options,
Readbacks menu.
Readbacks dialog box
There are three options for displaying system readbacks on the tune page:
• Readbacks displayed continuously.
• Readbacks hidden.
• Readbacks displayed only when differing from their defined values by
more than 10%.
A number of the readbacks are for diagnostic purposes only, their function
being to confirm a voltage is present. The acceptable variation between the set
value and the readback value varies depending on the particular tune
parameter. If concerned about any reading, contact the local service office for
advice.
Resetting the zero level 3-17

To change readback style:
1. Choose Readbacks from the tune page Options menu.
2. Select the readback style required.
3. Press OK.
Changing tune parameter settings

Most parameters can be modified in the following ways:
• Drag the slider bar using the mouse.
• Click on the slider bar and use the left and right arrow keys, to change
the value by one increment.
The edit window updates as the slider bar is activated.
• Type a new value into the edit window.
Other parameters have only an edit window, and are changed by direct
typing.
The speed with which the system responds to changes depends on the speed
with which the peak display refreshes. For the fastest response, set the Scope
Setup dialog box Scan Time (s) and Inter Scan Delay (s) values to be as short
as possible.
Source voltages
The following table lists the various components of Quattro micro API’s ion
optical system. The name in the table’s first column is the name used
throughout this manual to describe the component. When appropriate, the
second column shows the term used in the current MassLynx NT release.
Source voltages
Tune page
ESI+ve ESI-ve APcI+ve APcI-ve
name
ElectroSpray Capillary +3.0 (kV) -3.0 (kV) Not applicable
Probe
APcI Discharge Corona Not applicable 2 µA 2 µA
Pin
3-18 Tuning
Source voltages (Continued)
Tune page
ESI+ve ESI-ve APcI+ve APcI-ve
name
Sample Cone Cone +50 (V) -50 (V) +50 (V) -50 (V)
Extraction Cone Extractor +3 (V) -3 (V) +3 (V) -3 (V)
Hexapole RF Lens +0.2 (V) -0.2 (V) +0.2 (V) -0.2 (V)
The voltages shown are typical for an instrument in good condition. The
polarities given are those actually applied to the electrodes. Only positive
values need be entered via the tune page.

3-20 Tuning
4 Data Acquisition
Contents
Topic Page
Starting an acquisition 4-2
Monitoring an acquisition 4-8
Instrument data thresholds 4-10
System manager 4-16
Stopping an acquisition 4-16
The function list editor 4-16
4-1
Starting an acquisition
There are two ways of starting an acquisition:
• A single sample acquisition from the tune page
• A multiple sample acquisition from the MassLynx top level screen
Starting an acquisition from the tune page

• The easiest way to acquire data is directly from the tune page.
• Acquisitions can be started and stopped.
• Most of the scanning parameters can be controlled.
• Inlet programs cannot be used.
• Analog data cannot be acquired.
• Multiple sample sequences cannot be acquired.
To start a single sample acquisition:

1. Press Acquire on the tune page, or choose Acquire from the tune page
Window menu, the Start Acquisition dialog box will be invoked.
This will require changes to the settings to accommodate the required
mass range and scan times.
2. Press Start.
The Data File Name can be up to 128 characters. If the file already exists on
disk, a prompt is given to rename the file or to overwrite the existing one. The
file is written to the data directory of the current project.
4-2 Data Acquisition

Start Acquisition dialog box
To change the directory into which data are acquired:

1. Cancel the acquisition.
2. Create a new project by choosing Project Wizard, or open an existing one
by choosing Open Project, from the MassLynx top level file menu.
The Text area is used to enter the sample description. The description can be
displayed on any output of the acquired data and has a maximum length of
74 characters. To display text on more than one line, press CTRL+Return at
the end of a line.
The type of acquisition Function used to collect the data can be any of the
following:
• MS
• MS2
• Daughter
• Parent

• Neutral Loss
• Neutral Gain
More information is given on page 4-16.
The Data Format that are collected and stored on disk can be any of the
following:
• Centroid
• Continuum
• MCA
More information is given on these data formats later on in this chapter.
Set Mass specifies the mass (Daughter Mass, Parent Mass, etc.) that is used
for the particular function type. This control is disabled if the function
selected does not require a set mass.
Start Mass and End Mass specify the masses at which the scan starts and
stops. Start Mass must be lower than End Mass.
Run Duration is the length of the acquisition, measured in minutes.
Scan Time specifies the duration of each scan, in seconds.
Inter Scan Time specifies the time, in seconds, between a scan finishing and
the next one starting. During this period no data are stored.
Pressing Origin allows additional information about the sample to be
analyzed to be entered into the following fields:
• Submitter
• Job
• Task
• Conditions
Multiple samples
The MassLynx default page contains a sample list editor for defining multiple
samples which may be used together to perform a quantitative analysis. The
list of samples is set up using a spreadsheet style editor, which can be tailored
to suit different requirements.

To start a multi-sample acquisition:
1. Set up a sample list (see the MassLynx NT User’s Guide for details).
2. Choose Start from the top level Run menu, or press .

This invokes the Start Sample List Run dialog box.
Start Sample List Run dialog box
3. Check the Acquire Sample Data, Auto Process Samples and Auto
Quantify Samples boxes as required.
4. Enter values in the Run, From Sample and To Sample boxes.
The default is all samples in the list.
5. Check the Priority and/or Night Time Process boxes as required. See the
MassLynx NT User’s Guide for details.
6. Press OK.

7. Repeat the above procedure as required.
Sample lists are added to a queue and run sequentially unless Priority
or Night Time Process has been checked.
The sample which is currently being acquired has a next to it in the
sample list.
The Process controls allow processes to be run before and after the acquisition.
The Pre-Run control is used to specify the name of a process that is run before
acquisition of the files in the sample list.
The Post-Run control is used to specify the name of a process which is run
after acquisition of the files in the sample list. This could be used, for example,
to switch the instrument out of operate and to switch off various gases.
To run a process after each sample in the sample list has been acquired:
Format the sample list to display the Process column and enter the
name of the process to be run for each of the samples.
For the process to automatically operate on the data file which has just been
acquired:
Leave unchecked Use Acquired File as Default on the System tab of the
MassLynx Options dialog box.
The MassLynx Options dialog box is accessed by choosing Options from the
MassLynx Tools menu.
Automated quantification of sample list

To invoke the Quantify Samples dialog box:
1. Select Process Samples from the Quantify menu. Check the boxes
required and press OK.
The Quantify Samples dialog box allows automatic processing of data files
once they have been acquired. To perform integration, calibration of
standards, quantification of samples and printing of quantification reports
select the relevant check boxes. See the MassLynx NT User’s Guide for more
detailed information about using automated sample list analysis.
Integrate Samples integrates all the sample data files named in the peak list.

Calibrate Standards uses integration results to form quantify calibration
curves.
Quantify Samples uses integration results and quantify calibration curves to
calculate compound concentrations.
Quantify Samples dialog box
Print Quantify Reports produces hard copies of the results of integration and
quantification.
Export Results to LIMS produces a text file containing the quantification
results details for use with LIMS systems. If this box is checked, the LIMS
Export Browse button becomes enabled. Press Browse, select a file, or enter
the name of a new file, and press Save.
The Project field displays the project into which data are acquired.
To change the project into which data are acquired, the acquisition should be
canceled and a new project created by choosing Project Wizard, or an existing
project opened by choosing Open Project, from the MassLynx top level File
menu.
From Sample and To Sample set the range of samples in the sample list which
is analyzed.

Monitoring an acquisition
Acquisition status is shown on the MassLynx screen. The run time is shown
on the MS panel and the scan status, sample number and scan number are
shown on the Status bar at the bottom of the page.
The acquisition status (Scan Report) window

The acquisition status window, or Scan Report window, provides a scan by
scan statistical report of the progress of an acquisition.
Scan Report window
To invoke the Scan Report window:

1. Select Acquisition Status.
This shows details of the scan currently being acquired.

Chromatogram real-time update
To view in real time the chromatogram that is currently being acquired:
1. Open the data file using the MassLynx data browser.
2. Press , or select Real-Time Update from the Display menu. The

chromatogram display is updated as the acquisition proceeds.
Spectrum real-time update

To view in real time the spectrum that is currently being acquired:
1. Open the data file using the MassLynx data browser.
2. Press , or select Real-Time Update from the Display menu, the

Spectrum Real-Time Update dialog box is invoked.
Spectrum Real-Time Update dialog box
3. Select Enable Real-Time update.

Real-time update can also be turned on and off via the Real-Time
spectrum toolbar button.
Monitoring an acquisition 4-9

When real-time update is on, the display is continually updated with spectra
from the current acquisition. The actual information displayed is determined
by selecting one of the following radio buttons:
• Latest scan displays the last acquired scan. This is the default option.
• Average all scans updates the display with spectra formed by averaging
all the spectra that have so far been acquired.
• Average latest scans updates the display with spectra formed by
averaging the last n scans acquired, where n is specified in the
associated edit control.
Instrument data thresholds

MassLynx has several parameters that allow control over how the system
pre-processes data before it is sent to the host computer. These parameters
are contained in the Instrument Threshold Settings dialog box.
Instrument Threshold Settings dialog box
Instrument data thresholding allows the user to specify the type of data to
acquire and write to disk, and the type of data to discard and not write to disk.
Limiting the amount of data stored on disk can be particularly desirable when
acquiring continuum data and doing long LC runs.

To change data thresholding:
1. Choose Set Instrument Threshold from the tune page Options menu.
2. Make the required changes to the information.
3. Press OK.
These new parameters are downloaded at the start of the next acquisition
scan.
MaxEnt
The MaxEnt algorithm needs to measure noise accurately within a data file.
For this reason Ion Counting Threshold should be set to zero when acquiring
data to be analyzed using MaxEnt.
Profile data
The controls for profile data allow control of the amount of data collected
during a continuum data acquisition.
Baseline Level is used to lift or drop the baseline to see more or less of the
noise by positioning of the baseline above zero. Baseline Level is typically set
to a value of 0.
It is possible to use a negative baseline. This reduces the noise seen and acts
as a form of thresholding to be applied to 1/16 amu type samples. This takes
place after ion counting and therefore has a less significant effect than Ion
Counting Threshold.
To see more noise use a positive value.

Points per Dalton can have one of three values: 4, 8 or 16.
• Selecting 8 points instead of 16 results in data files approximately half
as big.
• Acquiring data at 16 points per Dalton gives the greatest possible
resolution.
• Acquiring data at 4 points per Dalton gives data with a smoothed
appearance.

Centroid data
Minimum centroid height sets a height below which detected peaks are
ignored. This reduces the size of acquired data files and is useful when
concentrating on larger peaks of interest. A suitable value can be arrived at by
inspecting spectral noise levels, and should be evaluated for each individual
system
Minimum points per peak is the minimum number of points that a continuum
peak must have to be centroided. A typical value is 10.
SIR data
SIR Baseline Level sets the position of the SIR baseline above zero. The
baseline level is typically set to 0. Increasing the value causes the baseline to
appear higher.
Ion counting threshold

Ion Counting Threshold sets the intensity level below which a data point is
ignored. This threshold is applied to all acquisitions, regardless of scanning
mode. It is also the most significant of all of the data manipulation variables
as it is applied to the raw data first.
When an acquisition is started the instrument performs a “prescan” with the
ion beam switched off so that the electronic noise level of the acquisition
system and its standard deviation can be measured. Ion Counting Threshold
only effects the electronic noise level of the system.
The Ion Counting Threshold level entered is multiplied by 1/10 of the
standard deviation of the noise to determine the intensity level to be used, so a
value of 10 equates to one standard deviation of the electronic noise level.
• Values can be set between 0 and 1000, the higher the number the more
data is discarded.
• If a value of zero is entered the intensity level is set so that it sits in the
middle of the noise which means that roughly half of the noise data is
acquired.
• A value of 10 places the threshold just above the noise so almost all of
the data is acquired.

• If a value of 200 is entered the threshold sits well above the noise level,
so very little noise data is acquired.
• A value of 30 is suitable for most data.
Ion Counting Threshold should be set so that background noise is removed
without significantly reducing the intensity of the smallest peaks of interest.
The table below shows the effects of changing baseline noise and ion counting
threshold on background noise and low intensity peaks.
Effects of changing baseline noise and ion counting threshold
Typical
Typical
Baseline Ion count Typical peak Typical saving on
background
level threshold profile intensity .DAT file
noise
size
0 0 0 0
1 0 0
2 0 0
5 0
0 10 4% 8%

Effects of changing baseline noise and ion counting threshold (Continued)
Typical
Typical
Baseline Ion count Typical peak Typical saving on
background
level threshold profile intensity .DAT file
noise
size
0 20 11% 10%
0 40 37% 61%
0 60 66% 69%
0 250 100% 83%
Profile data - spike removal

Spikes are distinguished from real data by the fact that the peaks are very
narrow and, when compared to their immediate neighbors, very intense. Data
points determined to be spikes are removed by setting the value of this data
point to the average of its immediate neighbors.
Spike removal involves some additional processing while acquiring, and
reduces the maximum achievable acquisition rates by approximately 30%.

To perform spike removal during an acquisition:
1. Check Use Spike Removal.
2. Refer to the tune page intensities to assess a suitable value for the
intensity threshold below which spikes are ignored. Set Minimum Spike
Intensity to this value.
3. A very low intensity signal may include single ion events that can be
combined to produce significant peaks. For this type of data Minimum
Spike Intensity should be set to a suitable value such that these single
ion events are not discarded as spikes.
4. Set a suitable value for Spike Percentage Ratio.
This ratio is used to determine if a data point is a spike by comparing
the data point to its immediate neighbors. For example, with Spike
Percentage Ratio set to 33%, a data point is regarded as a spike if its
intensity is three times (or more) greater than both its immediate
neighbors. A setting of 20% requires an intensity ratio of 5:1 to identify a
spike.
5. Press OK to accept any changes.
Any changes are not downloaded if Cancel is pressed.
Analog data
Select the number of samples to acquire per second from the drop-down list.

System manager
To check the communications between the MassLynx software and the

embedded PC:
1. Select Communications Status, the System Manager window is invoked.
System Manager window
Stopping an acquisition
To halt the acquisition:

1. From the tune page, press Stop.
2. From the MassLynx screen, choose Stop from the Run menu, or press
.
Data acquired up to this point is saved.
The function list editor
Introduction
The Function List Editor is used to set up the function(s) that the mass
spectrometer uses to scan the instrument during an acquisition. A function
list can be a mixture of different scanning techniques that can be arranged to
run either sequentially or concurrently during an acquisition.

To access the Function List Editor:
1. Press on the MS panel of the MassLynx screen.

Typical uses for mixed function acquisitions are to acquire different SIR
groups over different retention windows.
A function list is produced, saved on disk and then referenced by name when
an acquisition is started.
Function List Editor
The figure above shows a simple function list containing only one function: a
centroided mode full scan, between 500 and 1500 amu using ES+ ionization.
Immediately above the function bar display is a time scale that shows from
when the function is active, and for how long it runs. In this case, the function
starts after 5 minutes and then runs for 35 minutes, terminating after a total
elapsed time of 40 minutes.
A more complicated function list, with four SIR functions each running
sequentially for 5 minutes, is shown in the figure “Function list with four SIR
functions” on page 4-18.

The currently selected function is highlighted and enclosed in a rectangular
frame. If the display shows more than one function, a new function can be
selected either by clicking with the mouse, or by using the arrow keys on the
keyboard.
Function list with four SIR functions

The Function List Editor toolbar
The toolbar is displayed at the top of the tune window and allows some
common operations to be performed with a single click. The toolbar button
functions are shown in the table below.
Function List Editor toolbar buttons
Toolbar button Purpose

Create a new function list.
Open an existing function list.
Save the current function list to disk.
Print the current window in portrait format.
Edit the selected function.
Delete the selected function.
Move the selected function up the list of functions.
Move the selected function down the list of functions.
Create a new SIR function.
Create a new MRM function.
Create a new Full Scan function.
Create a new Parent function.

Function List Editor toolbar buttons (Continued)
Toolbar button Purpose

Create a new Daughter function.
Create a new Neutral Loss function.
Create a new Survey function.
Adding a new function

To add a new function to the list:
1. Click one of the toolbar buttons, or select the required function from the
Function menu.
The editor for the function type selected is invoked, showing default
values.
2. Make any changes required to the parameters and press OK to add the
new function.
The function editor for each scan type is discussed in detail later on in this
chapter.
Modifying an existing function

To modify an existing function:
1. Select the function in the function list.
2. Press , or double-click on the function.

This invokes the appropriate editor for the function type and allows
changes to be made.
The function list display is updated to show any changes.
Entering a new a value in Total Run Time and pressing sets the maximum
retention time for the experiment. The ratio of the functions defined is
maintained. For example, if two functions are defined one from 0 to 5 minutes

and the other 5 to 10 minutes then a Total Run Time of 10 minutes is
displayed. If this value is changed to 20, the first function now runs from 0 to
10 minutes and the second from 10 to 20 minutes.
Copying an existing function

To copy an existing function:
2. Select Copy and then Paste from the Edit menu.
3. Modify the parameters as described above.
Removing a function
To remove a function:
2. Press , choose Delete from the Edit menu, or press Del on the
3. When asked to confirm the deletion, select Yes.
Changing the order of functions

Functions are displayed in ascending Start Time and End Time order and this
order cannot be changed. For functions that have the same start and end time
the order in which they are performed can be changed.
To change the order of functions with same start and end time:
1. Highlight the required function.
2. Press or repeatedly until the function is in the required position.
Setting a solvent delay

To set a solvent delay for a function list:
1. Select Solvent Delay from the Options menu; the Solvent Delay dialog
box is invoked.

No data is stored during the solvent delay period, which means that solvent
peaks that would normally be seen eluting on the TIC chromatogram are no
longer seen.
For APcI functions, the APcI probe temperature is set to the value specified in
the APcI Probe Temp control for the period of the solvent delay.
To enable the divert/injector valve to be used as a divert valve, check Enable
Divert Valve. This diverts the flow of solvent during a solvent delay period
either to, or away from, the source for the time period shown in the solvent
delay timetable.
Up to four solvent delays can be programmed.
Solvent Delay dialog box
Analog channels
Up to four channels of analog data can be acquired, which are stored with the
data acquired from the mass spectrometer. Analog channels are typically used
to collect data from external units such as UV detectors, which must be
connected to the user input/output PCB as described on page 1-16.

A reading is made from the external channel at the end of each scan, and
stored with the data for that scan. The resolution of the chromatography for
an analog channel is therefore dependent on the scan speed used to acquire
the mass spectrometry data.
To access the Analog Data dialog box:

1. Select Analog Data from the Options menu on the Scan Functions dialog
box.
Analog Data dialog box
To store data for an analog channel:

1. Check the box(es) for the channel required.
2. Enter a textual description for each of the selected analog channels.
This description is used on the analog chromatogram dialog box as the
channel description. See the MassLynx NT User’s Guide.
3. Enter an Offset to align the external unit with the mass spectrometer.
4. Press OK.
Saving and restoring a function list

To save a function list:
1. Choose Save As from the function list File menu.
2. Enter a new file name, or select an existing file from the list displayed.
3. Press Save.

If the file already exists on disk, confirmation is requested to overwrite
the existing information.
4. Press Yes to overwrite the file, or No to select a different name.
When the editor is closed a prompt is issued to save any changed
function lists.
To restore a saved function list:

1. Choose Open from the function list File menu.
2. Select the name of the function list to open, either by typing its name or
by selecting it from the displayed list.
3. Press Open.
Setting up a full scan function

The full scan function editor is activated by pressing , or by
selecting MS Scan from the Functions menu, is used to set up centroid,
continuum and MCA functions.

Full scan function editor
Mass (m/z)
Start Mass and End Mass specify the masses at which the scan starts and
stops. Start Mass must be lower than End Mass.
Start Time and End Time specify the retention time, in minutes, during which
this function becomes active, and data are acquired.
Cone voltage
When Use Tune Page is checked, the cone voltage set on the tune page at the
start of the acquisition is used.
The cone voltage value cannot be altered during acquisition by typing new
values into the tune page, since the new values are not downloaded during
acquisition. This can only be done by acquiring from the tune page.

To apply a ramp to the cone voltage:
1. Check Use Cone Voltage Ramp and press CV Ramp to invoke the Cone
Ramp dialog box.
Cone Ramp dialog box
The four parameters define a gradient for the cone voltage which is then
extrapolated to cover the full mass range of the function.
Method
Ionization Mode specifies the ionization mode and polarity to be used during
acquisition.
Data specifies the type of data to be collected and stored on disk. There are
three options:
• Centroid stores data as centroided, intensity and mass assigned peaks.
Data are stored for every scan.
• Continuum. The signal received by the interface electronics is stored
regularly to give an analog intensity picture of the data being acquired.
Data are not centroided into peaks, but are stored for every scan.
Due to the fact that data are acquired to disk at all times, even when no
peaks are being acquired, data files tend to be significantly larger than
centroided ones.
It is possible, however, to set a threshold below which the data are not
stored. Depending on the nature of the data acquired, this can greatly
reduce these effects. The threshold can be set so that data considered to
be “noise” can be discarded, thus improving data acquisition speed and

reducing data file sizes. For more information about setting instrument
data thresholds see Instrument Data Thresholds, page.
• MCA (Multi Channel Analysis). MCA data can be thought of as
“summed continuum”, with only one intensity accumulated scan being
stored for a given experiment. As each scan is acquired, its intensity
data is added to the accumulated summed data of previous scans.
An advantage of MCA is that random noise does not accumulate as
rapidly as real data and therefore effectively averages out over a number
of scans. This emphasizes the real data and improves the signal-to-noise
ratio.
A further advantage of MCA is that, because data is written to disk only
at the end of an experiment, scanning speeds can be increased and
significantly less storage space is required.
The disadvantage of MCA is that, as there is only one scan, it cannot be
used for time-resolved data.
For MCA, Scans to Sum defines the number of scans to sum to create a
spectrum.
Scan duration (secs)

Scan Time specifies the duration of each scan in seconds, while Inter-Scan
Delay specifies the time, in seconds, between a scan finishing and the next one
starting. During this period no data are stored.
APcI probe
Probe Temp, in degrees centigrade, is enabled when Ionization Mode is set to
API.
When Use Tune Page Settings is selected, the APcI probe temperature set on
the tune page at the start of the acquisition is used. This control is enabled
when the Ionization Mode is set to API.
The APcI probe temperature value cannot be altered by typing new values
into the tune page during the acquisition, since the new values are not
downloaded during the acquisition. This can only be done by acquiring from
the tune page.

Setting up a SIR function
The SIR (Selected Ion Recording) technique is typically used in situations
where only a few specific masses are to be monitored. Since most of the data
acquisition time is spent on these masses, the technique is far more sensitive
than full scanning.
The SIR function editor is used to enter the masses to be monitored, along
with their dwell times, spans and inter-channel delay times.
SIR function editor
To set up a SIR function:
1. Press pressing , or select SIR from the Functions menu.

Many of the fields are described above for the Full Scan function editor. Only
those which differ are described below.

Channels
Up to 32 masses can be monitored.
To enter a mass:
1. Type suitable values into the Mass, Dwell and Cone boxes.
2. Press Add.
Dwell specifies the length of time in seconds for which the highlighted mass is
monitored.
To modify existing settings:

1. Double-click on a mass in the list.
This displays the values for the selected mass in the edit fields.
2. Change Mass, Dwell or Cone as required.
3. Press Change to update the values in the list.
To sort the list in order of ascending mass:

1. Press Sort.
Method
Inter Channel Delay specifies the time, in seconds, between finishing
monitoring the highlighted mass and starting monitoring the next mass in the
function.
Repeats is only relevant for experiments having more than one function and
specifies the number of repeats of the function.
Span specifies a small mass window applied centrally about the highlighted
mass. During acquisition this range is scanned over the specified Dwell time.
A span of zero can be set to simply “sit on” the specified mass.
Retention window
Start and End together specify the retention time, in minutes, during which
this function is active.

Setting up MS/MS scanning functions
MS/MS scanning function editors
Many of the fields in the MS/MS editors are similar to those in the Full Scan
function editor. Only fields which differ significantly are described below.

Mass
Daughter
This is the most commonly used MS/MS mode, and is used to look at
fragmentations of a particular ion. MS1 is set to the parent mass using
Daughters of, and is not scanned.
The resolution of MS1 can be lowered until the peak width at the base is two
masses wide without the daughter spectrum containing any ions from the
adjacent parent masses.
Start and End specify the mass range to be scanned by MS2.
It is possible to select the daughter mass to be greater than the parent
(precursor) mass. In this case, ions which have gained mass in the collision
cell, or are of higher mass-to-charge ratio, are detected. This can occur when a
multiply-charged ion fragments and loses a charge.
Parent
This mode is used to look for the parent of a particular fragment.
MS2 is set to the mass of the fragment, using Parents of, and is not scanned.
Start and End specify the mass range over which MS1 is scanned. Start is
normally set just below Parents of, and End to a value above the highest
expected parent mass.
There are often several masses from which a daughter may come, so that any
one fragment is derived from a number of different peaks.
MS2
In this mode MS2 is resolving, while MS1 transmits ions over a wide mass
range. While this scanning mode can be used for acquiring data it is mostly
used in the tune window, for setting and optimizing the acquisition conditions.
Neutral loss
In this mode, the peak in a spectrum that gives the neutral loss specified in
Loss of is detected. The precursor mass is scanned in MS1, and MS2 is
scanned at this mass less the neutral loss mass. Starting masses are therefore
detected on the mass scale of MS1. Start (for MS1) should be greater than
Loss of to give MS2 a valid start mass.
Neutral gain
This is an infrequently used mode, since the mass selected by MS2 is seldom
higher than that of MS1. It is applicable to studies where a precursor ion

gains mass by ion molecule reaction, or where multiply-charged ions fragment
into particles with a higher m/z value.
Collision energy
This specifies the collision energy, in electron volts, to be used for the collision
cell during the scan.
When Use Tune Page Settings is selected, the collision energy set on the tune
page is used. If it is required to adjust the setting during an acquisition then
the acquisition must be started from the tune page.
To apply a ramp to the collision energy:

1. Check Use Collision Energy Ramp.
2. Press CE Ramp to invoke the Collision Ramp dialog box.
Collision Ramp dialog box
The four parameters define values of collision energy for two particular
masses. This collision energy gradient is then extrapolated to cover the full
mass range of the function.
Setting up a MRM function

Multiple reaction monitoring (MRM) functions are set up in much the same
way as SIR functions, but allow a number of MS/MS transitions
(fragmentations) between MS1 and MS2 to be monitored.
All fields in the MRM function editor are similar to those already described.

MRM function editor
Setting up a survey function

Survey scans are used to search for precursor ions.
To access the dialog box:
1. Press pressing , or select Survey Scan from the Functions

menu in the scan functions editor.
The function list editor does not add survey functions to the list if non-survey
functions are present.
Survey and MSMS template pages

The Survey and MSMS Template pages allow the parameters to be set for MS
and MS/MS scanning during the survey, and are similar to normal function
editor pages.

Survey and MSMS Template Pages

MS to MS/MS switching
MS to MSMS page
Switch criteria
MS/MS scanning commences:
• If TIC is selected, and the TIC of the spectrum rises above the specified
Threshold.
• If Intensity is selected, and the intensity of the largest peak rises above
the specified Threshold.

When a peak top is found, no other peaks are looked for within the specified
Detection Window.
Number of Components is currently set to 1 and cannot be changed. The
number of non-coeluting precursors in a single run is not limited.
Precursor selection
If Automatic is selected all valid masses satisfying selection criteria are
monitored.
If Included masses only is selected, only masses in the Include List (see below)
are monitored.
If Included masses and Automatic is selected, masses on the Include List are
given priority. If no precursors are found, other valid masses are monitored.
A mass is valid if it is not on the Exclude List (see below), and it satisfies the
precursor selection criteria.
Detected precursor inclusion
Auto exclude and Always include are not currently available.
Include after time, if selected, allows a delay to be incorporated before
precursors are included.
Data
Discard uninteresting survey scans allows only the survey scans that detect
precursor ions to be stored. This saves on disk space, as survey scans that
contain no relevant data are rejected.

MS/MS to MS switching
MSMS to MS page
When MS/MS functions have been generated, they are carried out in parallel
until the conditions for switching to MS are satisfied.
When all MS/MS functions have stopped, the MS survey function is again
carried out.
Switch method
If the MS/MS to MS switch method is Default, the MS/MS function stops when
the MS/MS to MS switch criteria are met.

If the MS/MS to MS switch method is After Time, the MS/MS function stops
when the MS/MS to MS switch criteria are met, or otherwise, when the
specified time has elapsed.
Switch criteria
To define when MS scanning resumes:

1. Select one of the three conditions.
2. Set Threshold to a suitable value.
Including and excluding masses
Include/exclude masses page

Mass ranges and individual masses to be included or excluded from the
MS/MS scans are entered in the relevant Range boxes.
Masses on the Exclude list are not considered for detection.
Ranges take the form massX_massY.
Masses and ranges in a list are comma delimited, for example
100_200,202,236,250_300.
Monitoring acquisitions
When an acquisition is started, the Function Switching Status dialog box is
invoked, showing the precursors currently running.
Function Switching Status dialog box

5 Mass Calibration
Contents
Topic Page
Introduction 5-2
Overview 5-2
ElectroSpray 5-5
Atmospheric pressure chemical ionization (APcI) 5-29
5-1
Introduction
This chapter is divided into three sections:
• A brief general overview of the calibration process.
• A complete mass calibration of Quattro micro API using ElectroSpray
ionization with a mixture of sodium iodide and rubidium iodide as the
reference compound.
• A complete mass calibration of Quattro micro API using atmospheric
pressure chemical ionization (APcI) with PEG as the reference
compound.
See Chapter 8, for details of calibration solutions and their preparation.
Overview
MassLynx NT allows a fully automated mass calibration to be performed,
which covers the instrument for static and scanning modes of acquisition over
a variety of mass ranges and scanning speeds.
A mass spectrum of a reference compound (a calibration file) is acquired and
matched against a table of the expected masses of the peaks in the reference
compound which are stored as a reference file. The mass differences between
the reference peaks and calibration peaks are the calibration points. A
calibration curve is fitted through the calibration points.
The vertical distance of each calibration point from the curve is calculated.
This distance represents the remaining (or residual) mass difference after
calibration.
5-2 Mass Calibration

The standard deviation of the residuals is also calculated. This number is the
best single indication of the accuracy of the calibration.
Mass calibration
Overview 5-3
Calibration types
Each quadrupole analyser requires up to three calibration curves:
• A static calibration is used to “park” the analyser accurately on a specific
mass of interest (for example in tuning, SIR and MRM).
• A scanning calibration enables peaks acquired in a scanning acquisition
to be mass measured accurately.
• A scan speed compensation calibration compensates for “lag time” in the
system when the instrument is scanned rapidly.
A separate mass spectrum of the reference compound is acquired for each
selected calibration type.
Quattro micro API requires these three calibrations for both MS1 and MS2,
thereby generating a maximum of six calibration curves. The following table
shows which types of calibration are necessary for particular types of
experiment.
Calibration requirements
Calibration required
Experiment
MS1 MS2
MS All -
SIR Static -
MS/MS All All
MRM Static Static
The calibration process

• Tuning the instrument.
• Selecting the appropriate reference file for the reference sample to be
used.
• Starting an automatic calibration.
• Checking the calibration report.

ElectroSpray
Introduction
When a calibration is completed, it is possible to acquire data over any mass
range within the calibrated range. It is therefore sensible to calibrate over a
wide mass range.
With a mixture of sodium iodide and rubidium iodide, calibration over the
instrument’s full mass range is achievable.
Rubidium iodide spectrum
Preparing for calibration

Reference compound introduction
The example given here describes an automatic calibration which requires
reference compound to be present for several minutes. The introduction of the
reference compound is best achieved using the instrument’s syringe pump.
ElectroSpray 5-5
To introduce the reference compound:
1. Fill the syringe with the reference solution. See page 2-6.
2. Couple the syringe to the ElectroSpray Probe with fused silica tubing.
3. Set the pump to a flow rate of 10 µL/min.
Tuning
Before beginning calibration, and with reference solution admitted into the
source:
1. Set Multiplier to 650 V.
2. Adjust source parameters to optimize peak intensity and shape.
3. Set the resolution and ion energy parameters for unit mass resolution on
MS1 and MS2.
For a good peak distribution across the full mass range:

1. Check the intensity of some of the reference peaks above 1000 amu.
2. Check the intensity of the peak at m/z 173.
3. Ensure that no peaks are saturated on the tune page with a Gain of 1. If
necessary, reduce Multiplier or dilute the sample.
A cone voltage in the region of 45 is usually suitable.
Instrument threshold parameters

Before beginning the calibration procedure, some instrument parameters
must be checked.
For most low mass range calibrations, calibration data is acquired in
continuum mode.

To allow suitable scanning speeds to be used the continuum data
parameters need to be set correctly:
1. From the instrument tune page, select Options then Set Instrument
Threshold to invoke the Instrument Threshold Settings dialog box.
Instrument Threshold Settings dialog box
2. In the Profile Data section set Baseline Level to 0 and Points per Dalton
to 16.
3. Set Ion Counting Threshold and Spike Removal as appropriate, see
page 4-10.
4. Select OK to save the parameters.
ElectroSpray 5-7
Calibration options
To access the Calibration Options dialog box:
1. Select Calibration then Calibrate Instrument on the tune page.
Calibration Options dialog box
Selecting the reference file
To select the appropriate reference file:

1. Click on the arrow in the Reference File box and scroll through the files.
2. Select nairb.ref for a sodium iodide and rubidium iodide reference
solution.

Removing current calibrations
To remove the current calibration:

1. Select default.cal from the Calibrate menu option.
2. Save the changes to the default.cal file.
3. Check that there is no prior calibration associated with default.cal.
This ensures that a file with no calibration is currently active on the
instrument and prevents any previously saved calibrations from being
modified or overwritten.
Selecting parameters
Several parameters need to be set before a calibration is started. Default
parameters are set when the software is initially loaded which usually give a
suitable calibration, but under some conditions these may need to be adjusted.
Automatic calibration check

The Automatic Calibration Check dialog box is accessed from Edit, AutoCal
Check Parameters.
Automatic Calibration Check dialog box
ElectroSpray 5-9
This dialog box defines limits which the calibration must attain before the
instrument is successfully calibrated. Two user parameters can be set:
• Missed Reference Peaks sets the maximum number of consecutive peaks
that are not matched when comparing the reference spectrum and the
acquired calibration spectrum. The calibration fails if this number is
exceeded. The default value for this parameter, 2, is suitable in most
cases.
• Maximum Std Deviation is set to a default of 0.20. During calibration
the difference between the measured mass in the acquired calibration
file and the true mass in the reference file is taken for each pair of
matched peaks. If the standard deviation of the set of mass differences
exceeds the set value, the calibration fails. Reducing the value of the
standard deviation gives a more stringent limit. Increasing the standard
deviation means that the requirement is easier to meet, but this may
allow incorrect peak matching. Values greater than 0.20 should not be
used unless exceptional conditions are found.
Apply Span Correction should always be selected. This allows different mass
ranges to be scanned, within the calibrated range, without affecting mass
assignment.
Check Acquisition Calibration Ranges causes warning messages to be
displayed if an attempt is made to acquire data outside of the calibrated range
for mass and scan speed. It is advisable to leave selected.

Calibration parameters
These are accessed by selecting Edit, Calibration Parameters, this invokes the
Calibration Parameters dialog box.
Calibration Parameters dialog box
The Peak Match parameters determine the limits within which the acquired
data must lie for the software to recognize the calibration masses and result in
a successful calibration. The default values are shown in the figure above.
Increasing the Peak window and Initial error gives a greater chance of
incorrect peak matching. All peaks in the acquired spectrum below the
Intensity threshold value (measured as a percentage of the most intense peak
in the spectrum) are not used in the calibration procedure.
The Polynomial order of the curve has values from 1 to 5 as the available
options:
• A polynomial order of 1 should not be used.
• An order of 2 is suitable for wide mass ranges at the high end of the
mass scale, and for calibrating with widely spaced reference peaks.
Sodium iodide in particular has widely-spaced peaks (150 amu apart),
ElectroSpray 5-11
and horse heart myoglobin is used to calibrate higher up the mass scale,
so this is the recommended polynomial order for these calibrations.
• An order of 3 fits a cubic curve to the calibration.
• A fourth order is used for calibrations which include the lower end of the
mass scale, with closely spaced reference peaks. This is suitable for
calibrations with PEG which extend below 300 amu.
• A fifth order fit rarely has any benefit over a fourth order fit.
Mass measure parameters

These are accessed by selecting Edit, Mass Measure Parameters, this invokes
the Mass Measure dialog box.
Mass Measure dialog box

If continuum or MCA data are acquired for calibration, these parameters need
to be set before the calibration is carried out. If centroided data are used for
calibration, the mass measure parameters are not used.
With ElectroSpray calibrations, particularly with sodium iodide which has
some low intensity peaks at higher mass, it is recommended that continuum
or MCA data are acquired.
It is important that the data are smoothed correctly, and that the peak width
at half height (PWHH) is entered in the smoothing parameters as shown in
the above figure.
At high scan speeds, instrument resolution may decrease. Ensure that the
centroiding parameters are set to use the top of the peak so that mass
assignment of peaks is accurate.
Performing a calibration
Three types of calibration are available with MassLynx:
• Static Calibration
• Scanning Calibration
• Scan Speed Compensation
ElectroSpray 5-13
These are selected on the Automatic Calibration dialog box which is invoked
by selecting Start from the Calibrate dialog box.
Automatic Calibration dialog box
It is recommended that all three types of calibration are performed so that

mass ranges and scan speeds can be changed whilst maintaining correct mass
assignment. However, it is possible to have any combination of these
calibrations:
• If only a static calibration is present, the instrument is calibrated for
acquisitions where the quadrupoles are held at a single mass, as in SIR
or MRM.
• If only a scanning calibration is present, the instrument is only correctly
calibrated for scanning acquisitions over the same mass range and at
the same scan speed as those used for the calibration.
• If only a scan speed compensation is present (with no scanning
calibration having been performed), the scan speed compensation is
treated as a scanning calibration and the instrument is only correctly

calibrated for scanning acquisitions over the same mass range and at
the same scan speed as used for the calibration.
For the scan speed compensation to be used correctly, a scanning
calibration should also be performed.
• If static and scanning calibrations are both present, the instrument is
calibrated for acquisitions where the quadrupole is held at a single mass
and for scanning acquisitions with a mass range which lies within the
mass range of the scanning calibration, providing that the same scan
speed is used.
For example, if the instrument is calibrated from m/z 100 to 900 with a
2 s scan (400 amu/s), data can be acquired from 100 to 500 amu with a
1 s scan time (also 400 amu/s) whilst maintaining correct mass
assignment. In this case, the static calibration would be used to
determine the start mass of the acquisition and the scanning calibration
would be used for mass assignment and scan range.
• If scanning calibration and scan speed compensation are present then
the instrument is only calibrated for scanning acquisitions over the
same mass range as that used for the calibration, but the scan speed can
be changed, provided that it remains within the scan speeds used for the
two calibrations. The mass range should not be changed, as there is no
static calibration to locate the start mass.
• If all three types of calibration are present, all types of acquisition can be
used providing that the mass range and scan speed are between the
lower and upper limits used for the scanning calibration and the scan
speed compensation.
To perform a complete calibration:

1. Check the boxes in the Types area of the dialog box adjacent to Static
Calibration, Scanning Calibration and Scan Speed Compensation.
2. Check the MS1 and MS2 boxes.
3. In the Process area of the dialog box, check Acquire & Calibrate and
Print Report.
Acquisition parameters
Selecting Acquisition Parameters in the Automatic Calibration dialog box
invokes the Calibration Acquisition Setup dialog box, where the mass ranges,
scan speeds and acquisition mode are set. When this box is first accessed it
ElectroSpray 5-15
contains default parameters relevant to the chosen reference file. These
default parameters show the limits of scan range and scan speed for the
currently-selected instrument and calibration parameters.
Calibration Acquisition Setup dialog box
The upper area contains the Acquisition Parameters where mass range, run
time and data type are set.
When the instrument is fully calibrated, any mass range or scan speed is
allowed within the upper and lower limits dictated by the calibrations.
Select the nairb.ref file.
The solution described in Chapter 8 is suitable for use with this reference file.
If compatible reference solutions and reference files are used, then simply
selecting Default is sufficient action, no parameters need be entered
manually.
Run Duration sets the time spent acquiring data for each part of the
calibration. The time set must allow a minimum of three scans to be acquired
at the slowest scan speed used. Data are not acquired if the Run Duration is

too short. The slowest scan speed generally used is 100 amu/s. With Scan
From set to 20 amu and Scan To set to 2000 amu, a scan time of 19.8 s is
required, and an Inter Scan Delay (in the lower area of the box) of 0.1 s is
usually used. Therefore, the run duration must be greater than 59.6 seconds
(3 scans + 2 inter scan delays). A Run Duration of 1.00 minutes is suitable.
The lower area in the Calibration Acquisition Setup dialog box contains the
Scan Parameters.
When an instrument acquires data for a static calibration it examines the
reference file to find the expected reference masses, and then acquires data
over a small mass span around each peak’s expected position. Thus the
acquired data do not contain continuous scans. Each spectrum comprises
small regions of acquired data around each peak, separated by regions where
no data are acquired.
Static Span sets the size of this small region around each reference peak. A
span of 4.0 amu is typical.
Static Dwell determines how much time is spent acquiring data across the
span. A value of 0.1 s is suitable.
Slow Scan Time determines the scan speed used for the scanning calibration.
If both a scanning calibration and a scan speed compensation are to be
performed, the scan speed should be set to approximately 100 amu/s (a scan
time of 19.8 s over a mass range of 20 to 2000 amu). If only a scanning
calibration is to be performed (without scan speed compensation), the scan
speed should be set at the same speed to be used for later acquisitions.
Fast Scan Time determines the scan speed used for the scan speed
compensation, and the upper limit of scan speed that can be used for
subsequent acquisitions. A fast scan time of 4000 amu/s is adequate for most
applications. A scan range from 20 amu to 1100 amu requires a Fast Scan
Time of 0.26 s at an Inter Scan Delay of 0.1 s. The Acquisition Setup must be
edited to calibrate over the mass range desired.
Select OK to return to the Automatic Calibration dialog box.
Alternatively, select chosen values if a different calibration range is
required.
ElectroSpray 5-17
Starting the calibration process
To start the calibration process:

Select OK from the Automatic Calibration dialog box.
The instrument acquires all of the calibration files in the order, and
using the data file names, shown in the table below.
Calibration order and data files
Calibration Data file

MS1 static STATMS1
MS1 scanning SCNMS1
MS1 scan speed compensation FASTMS1
MS2 static STATMS2
MS2 scanning SCNMS2
MS2 scan speed compensation FASTMS2
Once all of the data have been acquired each data file is combined to give a
single spectrum which is then compared against the reference spectrum to
form a calibration. This process takes place in the same order as shown in the
table above. If the full calibration dialog box is open, a constantly updated
status message for the calibration is displayed.
If, when the process is completed, the calibration statistics meet with the
requirements specified by the selected calibration parameters, a successful
calibration message is displayed. A calibration report is then printed showing
a calibration curve for each of the calibration processes.
For the acquisition to be effective, it must be saved under a suitable file name.
Checking the calibration

The calibration (successful or failed) can be viewed in more detail by selecting
Process, Calibration From File from the Calibrate dialog box. The dialog box
which is then invoked allows the choice of calibration type for viewing. With
the required calibration selected, the correct calibration file is automatically
called up.

Scanning Calibration dialog box
Click Browse to select the calibration data file (for example STATMS1,
SCNMS1, FASTMS1, STATMS2 etc.). The selected file must be from the
appropriate project.
ElectroSpray 5-19
Clicking on OK repeats the calibration procedure for that particular file and
displays a calibration report.
Calibration report

The calibration report contains four displays:
• The acquired spectrum
• The reference spectrum
• A plot of mass difference against mass (the calibration curve)
• A plot of residual against mass
An expanded region can be displayed by clicking and dragging with the left
mouse button. In this way, the less intense peaks in the spectrum can be
examined to check that the correct peaks have been matched. The peaks in the
acquired spectrum which have been matched with a peak in the reference
spectrum are highlighted in a different color.
Calibration failure
If the calibration statistics do not meet the requirements, a message is
displayed describing at what point, and why, the calibration failed. This
message also states where the attempted calibration data can be viewed so
that the exact cause of failure can be determined.
Calibration failure message
There are a number of reasons for a calibration to fail:

No peaks
If the acquired calibration data file contains no peaks, the calibration fails.
This may be due to:
• Lack of reference compound.
• No flow of solvent into the source.
• Multiplier set too low.
ElectroSpray 5-21
Too many consecutive peaks missed
If the number of consecutive peaks which are not found exceeds the Missed
Reference Peaks parameter set in the Automatic Calibration Check dialog
box, the calibration fails. Peaks may be missed for the following reasons:
• The reference solution is running out so that the less intense peaks are
not detected.
• Multiplier is too low so that the less intense peaks are not detected.
• An incorrect ionization mode is selected. Check that the data have been
acquired with Ion Mode set to ES+.
Note that it is possible to calibrate in negative ion mode ElectroSpray
using the naineg.ref reference file with a suitable reference solution.
• Intensity threshold, set in the Calibration Parameters dialog box, is too
high. Peaks are present in the acquired calibration file but are ignored
because they are below the threshold level.
• Either Initial error or Peak window, set in the Calibration Parameters
dialog box, is too small. The calibration peaks lie outside the limits set
by these parameters.
• Maximum Std Deviation, set in the Automatic Calibration Check dialog
box, has been exceeded.
• The wrong reference file has been selected. Check that the correct file
(nairb.ref in this case) is selected in the Calibrate dialog box.
In the case of too many consecutive peaks missed:
• Check the data in the on-screen calibration report to see if the missed
peaks are present in the acquired calibration file.
• If the peaks are not present, the first three reasons (as explained above
in No peaks) are likely causes.
• If the peaks are present in the data, but are not recognized during
calibration then the latter four (Too many consecutive peaks missed) are
likely reasons.
Having taken the necessary action, proceed as follows:

1. If Intensity threshold, Initial error and Peak window are adjusted to
obtain a successful calibration, check the on-screen calibration report to
ensure that the correct peaks have been matched.

With a very low threshold and wide ranges set for the Initial error and
Peak window, it may be possible to select the wrong peaks and get a
“successful” calibration. This is particularly relevant for calibrations
with PEG, where there may be peaks due to PEG+H+, PEG+NH4+,
PEG+Na+, and also doubly-charged species.
2. Select OK from the calibration report window to accept the new
calibration, or select Cancel to retain the previous calibration.
Incorrect calibration
If the suggested calibration parameters are used, and providing that good
calibration data have been acquired, then the instrument should be calibrated
correctly. However in some circumstances it is possible to meet the calibration
criteria without matching the correct peaks. This situation is unusual, but it
is always sensible to examine the on-screen calibration report to check that
the correct peaks have been matched. These errors may occur when the
following parameters are set:
• Intensity threshold set to 0.
• Initial error too high (»2.0).
• Peak window too high (»1.5).
• Maximum Std Deviation too high (»0.2).
If the acquired spectrum looks like the reference spectrum and all of the
expected peaks are highlighted, the calibration is OK.
An alternative cause of incorrect calibration is from contamination or
background peaks. If a contamination or background peak lies within one of
the peak matching windows, and is more intense than the reference peak in
that window, then the wrong peak is selected. Under some conditions this may
happen with PEG. There are two ways to counter this:
• If the reference peak is closer to the centre of the peak window, the peak
window can be narrowed until the contamination peak is excluded. Take
care to ensure that no other reference peak is excluded.
• If the reference peak is not closer to the centre of the peak window, or if
by reducing the window other reference peaks are excluded, then the
calibration can be edited manually.
ElectroSpray 5-23
Manual editing of peak matching
If an incorrect peak has been matched in the calibration process, this peak can
be excluded manually from within the on-screen calibration report.
To manually edit Using the mouse, place the cursor over the peak in the
reference spectrum and click with the right mouse button.
Place the cursor over the peak in the acquired spectrum and click with
the right mouse button.
The peak is excluded and is no longer highlighted.
If the true reference peak is present, this can be included in the calibration by
the same procedure:
Place the cursor over the required peak and click with the right mouse
button.
The peak is matched with the closest peak in the reference spectrum.
Manually editing one peak does not affect the other matched peaks in the
calibration.
Saving the calibration

When the instrument is fully calibrated the calibration must be saved under a
file name so that it can be applied and recalled for future use.
The recalled calibration has the same constraints of mass range and scan
speed. The ion energy and resolution settings used for the calibration
acquisition are also recorded, as these can have an effect on mass assignment.
Verification
Once a full instrument calibration is in place it is not always necessary to
repeat the full calibration procedure when the instrument is next used.
Instead a calibration verification can be performed. (There is no benefit in
verifying each calibration individually, re-calibration is just as quick.)
If a scanning acquisition is to be made and the calibration is to be checked:

1. Set up the instrument and access the calibrate dialog box as though a
full calibration is to be carried out.
2. Set all peak matching parameters to the values that were used for the
calibration.

3. Invoke the Automatic Calibration dialog box by selecting Start on the
Calibrate dialog box.
Automatic Calibration dialog box
4. Select Scanning Calibration and deselect Static Calibration and Scan

Speed Compensation.
5. Deselect Acquire & Calibrate and select Acquire & Verify and Print
Report.
6. Select either MS1 or MS2, depending on the type of acquisition to be
performed.
7. Select Acquisition Parameters to call up the Calibration Acquisition
Set-up dialog box.
The parameters entered should be identical to the parameters originally
used for the calibration being verified.
ElectroSpray 5-25
8. Set Scan From, Scan To, Run Duration, Data Type, Scan Time and Inter
Scan Delay to agree with the acquisition parameters that are to be used
for data acquisition.
With only the Scanning Calibration selected, all of the other options in
this dialog box are unavailable.
9. Select OK to return to the previous dialog box, and OK again to start the
verification procedure.
A scanning acquisition is now performed. When the acquisition is complete,
the data are combined to give a single spectrum which is compared against
the reference file. A calibration curve is drawn and a report printed in a
similar way to when the original calibration was performed.
Unlike the original calibration procedure, the instrument calibration is not
changed and the report that is printed is a verification report.

Typical verification report
ElectroSpray calibration with PEG

Caution should be used when calibrating with PEG in ElectroSpray mode due
to the number of peaks which are produced. Although ammonium acetate is
added to the PEG reference solution to produce [M+NH4]+ ions, under some
conditions it is quite usual to see [M+H]+, [M+Na]+ and doubly-charged ions.
ElectroSpray 5-27
The spectrum shown in the figure below demonstrates how the PEG spectrum
can be dominated by doubly-charged ions (in this case [M+2NH4]2+) if the
wrong conditions are chosen. In this case, the concentration of ammonium
acetate in the reference solution is too high (5 mmol ammonium acetate is the
maximum that should be used) and Cone is too low.
ElectroSpray spectrum of PEG
A low Cone voltage encourages the production of doubly-charged ions. The

voltage should be at least 35 V.
Doubly-charged peaks can be identified, because the 13C isotope peak is
12
separated from the C isotope by only 0.5 Da/e. If the instrument is set to unit
mass, and data are acquired in continuum mode, the doubly-charged peaks
appear broader, as the isotopes are not resolved.

Atmospheric pressure chemical ionization (APcI)
Introduction
This section describes a complete mass calibration of Quattro micro API using
atmospheric pressure chemical ionization. The procedures described should be
followed only after reading “ElectroSpray” on page 5-5, describing the
automated calibration with ElectroSpray ionization.
Due to the high flow rates used with APcI, the residence time of an injection of
reference solution in the source is too short to allow a fully automated
calibration, and the procedure therefore has to be carried out in several steps.
The recommended reference compound for APcI is a solution of polyethylene
glycol (PEG) containing ammonium acetate. See Chapter 8, for advice on
preparing the reference solution. The figure “Typical APcI spectrum of PEG”
on page 5-30 shows a typical PEG + NH4+ spectrum.
With PEG, the possible calibration range is dependent upon the molecular
weight distribution of the PEGs used in the reference solution. For this
example PEG grades from PEG 200 to PEG 1000 are used.

Typical APcI spectrum of PEG
Preparing for calibration

Reference compound introduction
It is best to use a large volume injection loop (50 µl) with a solvent delivery
system set up to deliver 0.2 mL/min of 50:50 acetonitrile:water or
methanol:water through the injector and into the APcI source. An injection of
50 µL of reference solution lasts for approximately 15 s, allowing enough time
to perform a slow scanning calibration.

Tuning
Before beginning calibration:

1. Set Multiplier to 650 V.
2. Adjust the source and lens parameters to optimize peak intensity and
shape.
3. Set the resolution and ion energy parameters for unit mass resolution on
MS1 and MS2.
When a full calibration is completed, it is possible to acquire data over any
mass range within the calibrated range. It is therefore sensible to calibrate
over a wide mass range, and in this example the calibration covers up to
1050 amu.
Calibration options
To access the calibration options, click on Calibrate from the tune page.
To select the reference file:

1. Set pegnh4.ref as the reference file by clicking on the arrow in the
reference file box and scrolling through the files until the appropriate
file can be selected.
2. Leave the Use Air Refs box blank when calibrating in APcI.
To remove current calibrations:

1. Select Default from the Calibrate menu option.
2. Enter Yes to save the calibration into the most suitable project directory.
This ensures that a file with no calibration is currently active on the
instrument and prevents any previously saved calibrations from being
modified or overwritten.
Selecting calibration parameters

Several parameters need to be set before a calibration is started. Most of these
parameters can be set at the same value as for ElectroSpray. However, a
Polynomial order of 3 is recommended for the calibration Curve Fit.

Performing a calibration
The three types of calibration (Static, Scanning and Scan Speed) must be
carried out in single steps.
Static calibration
To perform a static calibration:

1. Access the Automatic Calibration dialog box by selecting Start from the
Calibrate page.
2. Check Static Calibration and MS1 in the Types area of the dialog box.
3. In the Process area of the dialog box, check Acquire & Calibrate.
Acquisition parameters
Selecting Acquisition Parameters invokes Calibration Acquisition Setup
dialog box; this contains the mass ranges, scan speeds and acquisition mode
relevant to the pegnh4.ref reference file.

Calibration Acquisition Setup dialog box
The upper area contains the Acquisition Parameters where mass range, run
time and data type are set. When the instrument is fully calibrated, any mass
range or scan speed is allowed within the upper and lower limits dictated by
the calibrations. It is therefore sensible to calibrate over a wide mass range.
Since the pegnh4.ref reference file has peaks from m/z 89 to m/z 2017, it is
possible to calibrate over this mass range. A calibration effective up to
1000 amu is sufficient for the majority of applications with APcI. The
following example shows a setup to achieve this.
Run Duration sets the time spent acquiring data for the static calibration. The
time set must allow chance to inject a volume of reference solution and acquire
several scans.
Data Type allows a choice of Centroided, Continuum or MCA data to be
acquired. For APcI, while either Continuum or Centroided data may be used,
Continuum is recommended.
The lower area in the Calibration Acquisition Setup dialog box contains the
Scan Parameters.

When an instrument acquires data for a static calibration it first examines the
selected reference file for the expected reference masses. It then acquires data
over a small mass span around the expected position of each peak. Thus the
acquired data do not contain continuous scans, but each “spectrum” is made
up of small regions of acquired data around each peak separated by blank
regions where no data are acquired.
Static Span sets the size of this small region around each reference peak. A
value of 4 amu is typical.
Static Dwell determines how much time is spent acquiring data across the
span. A value of 0.1 s is suitable.
Slow Scan Time and Fast Scan Time are not available when a static
calibration alone is selected.
Select OK from the Calibration Acquisition Setup dialog box to return to the
Automatic Calibration dialog box.
Acquiring data
To start the acquisition:

1. Select OK from the Automatic Calibration dialog box.
The instrument acquires a calibration file ready for static calibration
using the data file name STAT.
2. Inject the reference solution while data are being acquired.
Once the data have been acquired the instrument attempts to produce a static
calibration automatically. The data file contains only a few scans of the
reference compound, the remaining scans being of background.
As the automatic calibration procedure combines all of the scans in the data
file to produce a calibration spectrum, the resulting spectrum may be too weak
to give a successful calibration. Whether the calibration is successful or failed,
it is wise to check the calibration manually.
Manual calibration
To perform a manual calibration using the acquired data:

1. From the chromatogram window, open the calibration file STATMS1.
2. Determine the scan numbers at the beginning and end of the
chromatogram peak for the reference solution.

This can be achieved using Process, Combine Spectra and using the left
mouse button to drag across the peak. The start and end scans are
displayed in the Combine Spectra dialog box.
3. Return to the Calibrate dialog box. Access the manual calibration
options, in the Display Calibration Graphs dialog box, by selecting
Calibrate, From File.
Display Calibration Graphs dialog box
4. Select Static calibration type and MS1.

5. In the lower area, the data file STATMS1 should be selected
automatically. If this is not the case, the correct file can be selected by
clicking on Browse.
6. Enter the start and end scans of the reference data in the From and To
boxes.
7. Select OK to perform the calibration and display the calibration report
on the screen.

This report contains four displays:
• The acquired spectrum
• The reference spectrum
• A plot of mass difference against mass (the calibration curve)
• A plot of residual against mass
An expanded region can be displayed by clicking and dragging with the left
mouse button. In this way the less intense peaks in the spectrum can be
examined to check that the correct peaks have been matched. The peaks in the
acquired spectrum, which have been matched with a peak in the reference
spectrum, are highlighted in a different color.
Compare the acquired and reference spectra to ensure that the correct
peaks have been matched.
If insufficient peaks have been matched, or the wrong peaks have been
matched, refer to page 5-42.
If the correct peaks have been matched then the report can be printed out:
Select Print, Print from the report display.
To accept the calibration:

Select OK from the calibration report.

Calibration report - MS1 static

Calibration report - MS1 scanning

Calibration report - scan speed compensation

Scanning calibration and scan speed compensation
Acquiring data
To complete the calibration of the instrument, two further data files must be
acquired. Both files are acquired in scanning mode over the same mass range,
one at the slowest speed required for scanning acquisitions and one at the
fastest speed. Once these files have been acquired and used for calibration,
data may be acquired anywhere within the mass range at any scan speed
between the values used for the two sets of data. These data do not have to be
acquired through the Calibration dialog box, they can be acquired using the
normal scan setup and then accessed from the Calibration dialog box as
described below.
Scanning calibration
The recommended scan speed for the Scanning calibration is 100 amu/s.
1. Set Scan From to 50 amu and Scan To to 1050 amu.
2. Set Scan Time to 10 s and Inter Scan Delay to 0.1 s.
3. Select Continuum as the Data Type.
Although Continuum is recommended, Centroided data may be used.
4. Set Run Duration to 2.0 minutes.
This allows time to start the acquisition, inject the reference solution
and acquire several scans. With a solvent flow rate of 200 µL/min and a
50 µL loop in line, an injection of reference solution lasts approximately
15 s, allowing at least one full scan of useful data to be acquired.
5. Choose a filename for the data.
The filename SCNMS1, the name used during an automatic calibration,
is valid.
6. Start the acquisition and inject the reference solution.
Scan speed compensation

The recommended scan speed for the scan speed compensation is 4000 amu/s.
1. Set Scan From to 50 amu and Scan To to 1050 amu.
2. Set Scan Time to 0.24 s and Inter Scan Delay to 0.1 s.
3. Select Continuum as the Data Type.

Although Continuum is recommended, Centroided data may be used. It
is possible to scan more quickly in Centroided mode, but it is unlikely
that a faster acquisition rate would be needed for general use.
4. Set Run Duration to 2.0 minutes.
5. Choose a filename for the data.
The filename FASTMS1, the name used during an automatic
calibration, is valid.
6. Start the acquisition and inject the reference solution.
Manual calibration
To begin manual calibration:

1. Find the start and end scans of the reference data for each file in the
same way as for the static calibration file.
2. From the tune page select Calibration.
3. Select Scanning calibration type and MS1, Calibrate Instrument,
Calibrate From File.
4. In the lower area, the data filename SCNMS1 should be selected
automatically. If this is not the case, or if an alternative filename has
been used for the slow scanning acquisition, the correct file can be
selected by clicking on Browse.
5. Enter the start and end scans of the reference data in the From and To
boxes.
6. Select OK to perform the calibration and display the calibration report
on the screen in a similar way to the Static calibration.
7. Compare the acquired and reference spectra to ensure that the correct
peaks have been matched.
If the correct peaks have been matched, the calibration report can be printed
out:
Select Print, OK from the report display.
If insufficient peaks have been matched or the wrong peaks have been
matched see page 5-42.

To accept the calibration:
Select OK from the calibration report.
The same procedure is used for the Scan Speed Compensation except that
Scan Speed Compensation is selected in the dialog box, and the fast scanning
file is used. Note that for the Scan Speed Compensation, the default file is
FASTMS1. If an alternative filename has been used, this must be selected
using the data browser.
Once all three calibrations (Static, Scanning and Scan Speed Compensation)
have been completed, the instrument can be used for any mass range within
the limits of the scanning calibrations, and at any scan speed from 100 to
4000 amu/s.
Calibrating MS2
The calibration of MS2 is carried out in exactly the same manner as above,
except that data is acquired in MS2 mode instead of MS1.
Using the instrument
Once all six calibrations (Static, Scanning and Scan Speed Compensation,
each for both MS1 and MS2) have been completed, the instrument can be used
for any mass range within the limits of the scanning calibrations and at any
scan speed from 100 to 4000 amu/s.
Calibration failure
When calibration is performed manually there is no warning message to show
that the calibration has not met the set criteria. This must be judged by
viewing the on-screen calibration report and examining the matched peaks
and statistics associated with the report. There are a number of reasons for a
calibration to fail:
No peaks
If the acquired calibration data file contains no peaks the calibration has
failed. This may be due to:
• Lack of reference compound.
• Wrong scans or wrong data file being used for the calibration.
• No flow of solvent into the source.
• Multiplier set too low.

Too many consecutive peaks missed
If the number of consecutive peaks which are not found exceeds the limit set
in the Automatic Calibration Check dialog box, the calibration has failed.
Peaks may be missed for the following reasons:
• The reference solution is running out, causing less intense peaks to not
be detected.
• Multiplier is too low and less intense peaks are not detected.
• The incorrect ionization mode is selected. Check that the data has been
acquired with Ion Mode set to APcI+.
• Intensity threshold, set in the Calibration Parameters dialog box, is too
high. Peaks are present in the acquired calibration file but are ignored
because they are below the threshold level.
• Either Initial error or Peak window, set in the Calibration Parameters
dialog box, is too small. The calibration peaks lie outside the limits set
by these parameters.
• Maximum Std Deviation, set in the Automatic Calibration Check dialog
box, has been exceeded.
• The wrong reference file has been selected. Check that the correct file
(pegNH4.ref in this case) is selected in the Calibrate dialog box.
In the case of too many consecutive peaks missed:
• Check the on-screen calibration report to see if the missed peaks are
present in the acquired calibration file.
• If the peaks are not present, the first four reasons above (No peaks) are
likely causes.
• If the peaks are present in the data, but are not recognized during
calibration, then the latter four reasons (Too many consecutive peaks
missed) are likely reasons.
Having taken the necessary action, proceed as follows:

1. If Intensity threshold, Initial error and Peak window are adjusted to
obtain a successful calibration, check the on-screen calibration report to
ensure that the correct peaks have been matched.
With a very low threshold and wide ranges set for the Initial error and
Peak window, it may be possible to select the wrong peaks and get a
“successful” calibration. This is particularly relevant for calibrations

with PEG, where there may be peaks due to PEG+H+, PEG+NH4+ and
PEG+Na. This situation is unusual, but it is always wise to examine the
on-screen calibration report to check that the correct peaks have been
matched.
2. Select OK from the calibration report window to accept the new
calibration, or select Cancel to retain the previous calibration.
Incorrect calibration
If the suggested calibration parameters are used, and providing that good
calibration data have been acquired, the instrument normally calibrates
correctly. However, in some circumstances it is possible to meet the
calibration criteria without matching the correct peaks.
This situation is unusual, but it is always wise to examine the on-screen
calibration report to check that the correct peaks have been matched. These
errors may occur when the following parameters are set:
• Intensity threshold set to 0
• Initial error too high (>2.0)
• Peak window too high (>1.5)
• Maximum Std Deviation too high >0.2).
If the acquired spectrum looks like the reference spectrum and all of the
expected peaks are highlighted then the calibration is OK.
An alternative cause of calibration failure is from contamination or
background peaks. If a contamination or background peak lies within one of
the peak matching windows, and is more intense than the reference peak in
that window, then the wrong peak is selected. Under some conditions this may
happen with PEG. There are two ways to counter this:
• If the reference peak is closer to the centre of the peak window, the peak
window can be narrowed until the contamination peak is excluded. Take
care to ensure that no other reference peak is excluded.
• If the reference peak is not closer to the centre of the peak window, or if
by reducing the window other reference peaks are excluded, the
calibration can be edited manually.

Manual editing of peak matching
If an incorrect peak has been matched in the calibration process, this peak can
be excluded manually from within the on-screen calibration report.
To manually edit peak matching:

1. Using the mouse, place the cursor over the peak in the reference
spectrum and click with the right mouse button.
2. Place the cursor over the peak in the acquired spectrum and click with
the right mouse button.
The peak is excluded and is no longer highlighted.
If the true reference peak is present, this can be included in the calibration by
the same procedure:
Place the cursor over the required peak and click with the right mouse
button.
The peak is matched with the closest peak in the reference spectrum.
Manually editing one peak does not affect the other matched peaks in the
calibration.
Saving the calibration

When the instrument is fully calibrated the calibration, for it to be effective,
must be saved under a filename so that it can be recalled for future use. For
example, it is possible to save calibrations for use with different ionization
modes, so that when an ionization source is switched the corresponding
calibration is recalled.
The recalled calibration has the same constraints of mass range and scan
speed. The ion energy and resolution settings used for the calibration
acquisition are also recorded as these can have an effect on mass assignment.

Manual verification
Once a full instrument calibration is in place it is not always necessary to
repeat the full calibration procedure when the instrument is next used.
Instead a calibration verification can be performed. (There is no benefit in
verifying each calibration individually, re-calibration is just as quick.)
If a scanning acquisition is to be made and the calibration is to be checked:

1. Set up a scanning acquisition over the required mass range and at the
required scan speed in the normal way.
2. Start the acquisition and inject the reference solution so that reference
data is acquired.
3. Stop the acquisition.
4. Invoke the Calibrate dialog box and set all peak matching parameters to
the same values that were used for the calibration.
5. Select Process, Verification from file, the Display Verification Graphs
dialog box is invoked.
6. Select Scanning Calibration and either MS1 or MS2 depending on the
type of data acquired.
7. Click on Browse, select the acquired file and enter the start and end
scans of the reference data.
8. Select OK to verify the calibration.

A calibration curve is produced and displayed on the screen in a similar
way to when the original calibration was performed. An example is
shown in the figure “Verification report” on page 5-48.
Display Verification Graphs dialog box

Verification report
When OK is selected from this report, unlike the original calibration

procedure, the instrument calibration is not changed. As the verification
procedure uses the same matching parameters as the calibration procedure, it
is possible to validate the current calibration without re-calibrating the
instrument.
The report can be printed out by selecting Print, OK from the verify report.

6 Maintenance Procedures
Contents
Topic Page
Maintenance schedule 6-2
Safety and handling 6-3
Routine maintenance 6-4
Replacing parts 6-28
6-1
Maintenance schedule
The following table lists periodic maintenance schedules to be followed to
ensure optimum performance.
The maintenance frequencies shown apply to instruments that normally
receive moderate use.
Maintenance schedule
Maintenance procedure Frequency

Gas-ballast the rotary pump Daily (APcI)
Weekly (ESI)
Check and adjust the rotary Weekly
pump oil level
Change the rotary pump oil Every 3000 hours of pump operation
Clean the cone gas cone, sample When sensitivity decreases to
cone, and baffle plate unacceptable levels
Clean the ESI and APcI probe When sensitivity decreases to
tip unacceptable levels
Clean the corona discharge When sensitivity decreases to
needle (APcI mode) unacceptable levels
Clean the ion block assembly When it is visibly fouled
When background or high peak
contaminants are unacceptably high
Clean all source components When sensitivity decreases to
unacceptable levels
When cleaning the cone gas cone, sample
cone, and baffle plate fails to improve
analytical results
Clean the ion block assembly When it is visibly fouled
When background or high peak
contaminants are unacceptably high
Clean all source components When sensitivity decreases to
unacceptable levels
When cleaning the cone gas cone, sample
cone, and baffle plate fails to improve
analytical results
6-2 Maintenance Procedures

Safety and handling
Bear in mind the following safety considerations when performing
maintenance procedures.
Warning: Never open the instrument’s top or side panels to access power
supplies or other components. The instrument does not contain
user-serviceable parts.
Warning: Observe good laboratory practice when handling solvents,

changing tubing, or operating the Quattro micro API. Know the physical
and chemical properties of the solvents to be used. Refer to their
Material Safety Data Sheets
Caution:
• Never disconnect an electrical assembly while the system is plugged
in. This could damage electrical parts. Also, turn off the power and
wait 10 seconds before disconnecting an assembly.
• Do not touch integrated circuit chips or other circuit board
components. Static electric charge can damage electronic
components.
Proper operating procedures

Follow all recommended procedures and guidelines to maintain the detector’s
operating efficiency.
Maintenance equipment
Routine parts cleaning requires the following equipment:
• An ultrasonic bath with a minimum chamber size of
12 inches × 6 inches × 4 inches (300 mm × 150 mm × 100 mm).
• Glass vessels, approximately 4 inches (100 mm) in diameter and
4.25 inches (120 mm) high.
• A 500-mL graduated cylinder (to use when cleaning the hexapole
assembly).

Routine maintenance
Gas-ballasting the rotary pump

When the rotary pump draws large quantities of solvent vapors, the vapors
tend to condense in the pump oil, reducing pump efficiency. Gas-ballasting
purges condensed contaminants from the oil and returns any oil to the pump
from the oil mist filter. Gas-ballast the rotary pump when the following
conditions apply:
• With ESI operation, once a week.
• With frequent APcI operation, once a day.
• If the pump oil appears cloudy.
• If the vacuum pressure is higher than normal.
• If condensate forms in the rotary pump exhaust line.
• When changing the rotary pump oil.
• If the level of accumulated oil in the oil mist filter is high.
Caution:
• Failure to routinely gas-ballast the rotary pump shortens oil life and
consequently shortens pump life.
• Do not vent the instrument when the rotary pump is gas-ballasting.
• Do not gas-ballast the rotary pump while Quattro micro API is in the
Operate mode.
• Never gas-ballast the rotary pump for more than 2 hours.
To gas-ballast the pump:

1. Shut the vacuum system isolation valve, moving its handle fully to the
right.
2. Turn on the gas-ballast.

Rotary pump
3. When the oil is clear and has drained back to the rotary pump, return
the gas-ballast control to its normal position.
4. Open the vacuum system isolation valve.
Checking the rotary pump oil

The oil level can be checked while the pump is operating. However, the
instrument must be vented and shut down before adding oil.
The rotary pump oil level appears in the oil level sight glass on the pump.
Check the oil level at weekly intervals; at all times it should be at, or near, the
MAX level as indicated by the markings beside the sight glass. If oil must be
added, vent and shut down the Quattro micro API before removing the oil
filler plug.

Examine the oil each time the oil level is checked. It should be colorless and
free of visible contaminants. If the oil is discolored, change it as described on
page 6-6.
Changing the rotary pump oil

Change the rotary pump oil every 3 to 4 months, or whenever it becomes
noticeably discolored.
Required materials
• Rubber gloves
• Flat-blade screwdriver
• Container to catch used oil
• Funnel
• Vacuum oil (use only Ultragrade 19 or Inland Q45 (Edwards 45) vacuum
pump oil)
Procedure
To change the rotary pump oil:

1. Operate the pump to warm the oil before draining.
2. Gas-ballast the rotary pump as described on page 6-4.
3. Vent and shut down the instrument, turning the power switch to off.
4. Raise the pump 6 to 8 inches (150 to 200 mm) above the floor, if
necessary.
5. Place an object under the motor to tilt the pump toward the side on
which the oil drain plug is located (see the figure “Rotary pump” on
page 6-5).
6. Remove the oil filler plug to facilitate drainage.
7. Using the flat-blade screwdriver, remove the oil drain plug.
8. Let the oil drain completely, then refit the oil drain plug.
9. Fill the pump until the oil in the sight glass reaches the MAX level.
10. Allow a few minutes for the oil to drain into the pump.

11. Recheck the oil level, and add more oil if necessary.
12. Refit the oil filler plug and, if necessary, lower the pump to the floor.
13. Turn the rotary pump power switch to On.
Cleaning the source assembly

Overview
The sample cone, cone gas cone, and baffle plate should be cleaned either:
• When they are visibly fouled.
Or:
• When LC and sample-related causes for decreased signal intensity have
been ruled out.
When cleaning these parts fails to increase signal sensitivity, also clean the
extraction lens, hexapole, and ion block.
The source-cleaning procedure is as follows:

1. Disassemble the source components, which are fully described later in
this chapter.
2. Clean the source components.
3. Remove and clean the hexapole assembly.
4. Refit the hexapole assembly.
5. Reassemble the source components.
Required materials
The following materials are required to clean the source components:
• Lint-free cotton or powder-free nitrile gloves
• 6-inch (150-mm) or needle-nose pliers
• 2.5-mm hex wrench
• 6-mm hex wrench
• Small, flat-blade screwdriver
• Large, flat-blade screwdriver

• Clean 1000-mL beaker
• Clean 500-mL graduated cylinder
Use only glassware not previously cleaned with surfactants.
• HPLC-grade methanol
• HPLC-grade water
• Formic acid
• Ultrasonic bath
• Source of oil-free, inert gas (nitrogen or helium) for drying (air-drying
optional).
• Lint-free paper towels
Spare parts
The following spare parts may be required when cleaning source components:
• Ion block D-ring (AS035)
• Viton O-ring (AS214)
• Extraction cone O-ring
• Sample cone O-ring
Disassembling the source components
To disassemble the source components:

1. Set Source Temp and either APcI Probe Temp or Desolvation Temp to
20 °C, to switch off the heaters.
Caution: The probe should be cooled to below 100 °C and the

source cooled to below 50 °C before removal. Failure to do so will
shorten the life of the probe heater.
2. To reduce cooling time significantly, continue flowing API gas.
3. When the probe has cooled, stop the nitrogen flow by deselecting on
the toolbar or by choosing Gas from the Gas menu.
4. Stop the liquid flow, and disconnect the LC line from the probe.
5. Click Press For Standby on the MassLynx tune page.

6. The icon changes from green to red. This means all high voltages are
turned off, as well as the ESI desolvation/APcI probe heater.
7. Select Options then Vent from the tune page, and click OK when the
message box appears. If cleaning only the sample and cone gas cones, it
is not necessary to vent the system.
8. Select Options then Vent from the tune page, and click OK when the
message box appears. If cleaning only the sample and cone gas cones, it
is not necessary to vent the system.
When the instrument is vented, power to the turbomolecular pump is
interrupted. However the pump does not stop immediately. Venting is a
controlled and safe process over several minutes designed to prevent
damage to the instrument. When the turbomolecular pump stops, a vent
valve opens automatically and vents the vacuum chamber to the
atmosphere. The rotary pump stops running about 3 minutes after the
vent cycle begins.
9. Disconnect the front panel gas and electrical connections.
Warning: Handle the probe carefully. It might still be hot.
10. Unscrew the probe’s two knurled thumbscrews and retract it from the
source.

Probe and source connections
Probe lead
Desolvation gas Knurled

thumbscrew
Nebulizer gas
Probe
Probe adjuster
plate
Source enclosure
door
Source enclosure door

securing clip
11. Remove the center section panel from the front of the instrument,
pulling it away from the instrument.

Removable center panel
Removable
Center Panel
12. Unfasten the source enclosure door’s securing clips and open the door.
Caution:
• Wear lint-free cotton or powder-free nitrile gloves for the rest of
the cleaning procedure.
• Take care not to scratch the highly polished cone orifice
surfaces.
13. If using an APcI probe, carefully remove the corona discharge needle.

Source enclosure
Source enclosure
securing screw
Gas cone
6-mm hex screw

Corona discharge
needle
PTFE tubing
14. Remove the PTFE tube attached to the cone gas cone.
15. Remove the two screws that secure the cone retainer, using the small,
flat-blade screwdriver.

Ion block
6-mm Hex
Screw Isolation
Valve
Cone
Retainer
16. Remove the cone gas cone, O-ring, and sample cone from the ion block.
Removing the sample cone
Isolation
Valve
Sample
Cone
Cone Gas
Cone
Cone
Retainer Clip
17. Carefully separate the sample cone from the gas cone, then remove the
sample cone O-ring.
18. Remove the baffle plate, and set all pieces aside.

19. Remove the two screws that secure the ion block, using the 6-mm hex
wrench.
20. Remove the ion block from the ion block support.
Exploded diagram
PEEK connector block
Pumping block O-ring
PEEK ion block

support
Isolation valve
PTFE ring
O-rings
O-rings
Extraction cone
Cone gas cone

Cone retainer
21. Place the ion block on a flat surface, and remove any O-rings.
22. Use a screwdriver to remove the hold-down screw from the PEEK
extraction cone retainer.

Removing the extraction cone
Hold-Down
Screw PEEK Extraction
Extraction
Cone Retainer
Cone
23. Place the shaft of a small, flat-blade screwdriver in the ion block relief,
and carefully pry the insulator O-ring away from the ion block.
24. Take care not to damage the ion block surface and insulator O-ring.
25. Grasp the extraction cone pin with the needle-nose pliers, then lift the
extraction cone from the ion block.
26. Insert the small, flat-blade screwdriver under the inner edge of
polymeric seal ring and carefully pry the seal ring and O-ring out of the
ion block.
27. Take care not to damage the seal and O-ring on the ion block.
28. Remove the D-shaped O-ring from the front of the ion block.
29. Remove the ion block plug and seal using a flat-blade screwdriver.
30. Remove the O-ring from around the sample cone orifice, taking care not
to scratch the ion block surface.

Cleaning the source components
To clean the source components:

1. Place the ion block in a beaker with methanol:water (1:1).
2. Place the beaker containing the ion block and methanol:water mixture
in an ultrasonic bath for 20 minutes.
3. Remove the ion block from the methanol:water mixture, and place it in a
beaker containing 100% methanol.
Warning: Use extreme care when working with formic acid. Use a
fume hood and appropriate protective equipment.
4. If the sample cone contains debris, place a drop of formic acid on its
orifice.
5. Place the sample cone, cone gas cone, and extraction cone in a beaker
with methanol:water (1:1).
6. If the parts are obviously fouled, use a mixture of 45% methanol, 45%
water, and 10% formic acid.
7. Expose all parts to ultrasound for about 30 minutes.
8. If formic acid was used in the cleaning solution:
a. Rinse the parts, immersing them in a beaker of water and setting
the beaker in an ultrasonic bath for about 20 minutes to remove
formic acid from them.
b. Displace the water by immersing the parts in a beaker of methanol
and setting the beaker in the ultrasonic bath for 10 minutes.
9. Carefully remove the parts from the beaker and blow-dry them, using
inert, oil-free gas.
Alternatively. the parts may be placed on lint-free towels and allowed to
air dry. Wipe off any water spots with a lint-free cloth.

Removing and cleaning the hexapole assembly
To remove and clean the hexapole assembly:

1. Remove the screws securing the ion block support with the 3 mm hex
wrench, and remove the ion block support from the pumping block.
Removing the hexapole
Pumping block
O-rings
PEEK ion block
support
Hexapole
assembly
2. Remove any O-rings that remain stuck to the surface of the pumping
block.
Caution: Never squeeze the hexapole rods together when

removing the hexapole, as their orientation relative to one another
is critical to the Quattro micro API’s performance. Take care not to
scratch the bored surfaces of the pumping block as the hexapole is
withdrawn.
3. Grasp the hexapole gently by hand, and carefully slide it out.

4. Bend a length of stainless steel tubing into a hook shape, and insert the
hook into one of the holes in the rear support ring.

Hexapole assembly
Rear
Support
Ring
Alignment
Notch
5. Carefully suspend the hexapole assembly in a graduated cylinder, then

add methanol to the cylinder until the assembly is covered.
6. Place the graduated cylinder in an ultrasonic bath for 30 minutes.
7. Remove the hexapole assembly from the graduated cylinder, and place it
on a lint-free cloth. Allow it to air-dry, or use a nitrogen flow to dry it.
8. Insert the hexapole assembly by aligning the notches in the differential
aperture at the rear of the hexapole with the two bottom support rails on
the analyser assembly, then carefully slide it into place. Be sure to insert
the assembly fully.
9. Check the condition of the three rear ion block support O-rings,
replacing them with new ones if necessary. Ensure that the O-rings are
properly installed before reattaching the ion block support. Also make
sure the pin in the ion block support aligns with the notch on the ion
block.
10. Secure by alternately tightening the retaining screws.
Reassembling the source components
To reassemble the source components:

1. Check the condition of the two front ion block support O-rings. Replace
them with new O-rings, if necessary. Be sure they are properly installed
before proceeding.
®
2. Refit the Vespel sealing ring and O-ring on the ion block support.

3. Press the extraction cone into place in the ion block support, and secure
it with the PEEK retainer and screw.
4. Refit the sample cone and its O-ring, then the cone gas cone, secured
with the retainer spring and two screws as well as the ion block plug and
seal.
5. Refit the ion block assembly onto the PEEK support block, then the cone
gas cone secured with two 6-mm hex screws. Avoid overtightening.
6. If only the sample and cone gas cone have been cleaned, turn the
isolation valve back to Open.
7. If using APcI, refit the corona discharge needle (see page 6-17).
8. Reattach the PTFE tube to the cone gas cone.
9. Close the source enclosure door, and fasten the door’s securing clips.
10. Pump down the instrument and turn on the API gas.
Cleaning and replacing the corona discharge needle

The corona discharge needle should be cleaned if it looks corroded or black, or
when the signal intensity weakens.
Required materials
The following materials are needed to clean the corona discharge needle:
• Lint-free cotton or powder-free nitrile gloves
• Lapping film
• Lint-free tissue
Procedure
To remove, clean and replace the corona discharge needle:

1. Set Source Temp and APcI Probe Temp to 20 °C to switch off the
heaters.

To reduce cooling time significantly, continue flowing API gas.
Caution: Do not remove the probe before it cools to below 100 °C

and the source heater cools to below 50 °C. Doing so will shorten
the life of the probe heater.
2. When the probe has cooled, stop the nitrogen flow by deselecting on
the toolbar or by choosing Gas from the Gas menu.
3. Click Press For Standby on the MassLynx tune page.
4. Ensure that the icon changes from green to red. This means all high
voltages are turned off, as well as the APcI probe heater.
5. Remove the center panel from the front of the instrument.
6. Disconnect the nebulizer gas and the probe electrical connection.
Probe and source connections
Probe lead
Desolvation gas Knurled

thumbscrews (2)
Nebulizer gas
Probe
Probe adjuster
plate
Source enclosure
door

securing clips (2)

7. Remove the two knurled thumbscrews from the top of the probe.
8. Remove the APcI probe.
Warning: The inner surfaces of the source enclosure and its

constituent components are hot.
9. Unfasten the source enclosure door’s securing clips, and open the door.
Caution: Wear lint-free cotton or powder-free nitrile gloves for the

rest of the cleaning procedure.
10. Remove the corona discharge needle from the source, pulling it straight
out.
Source enclosure
Source enclosure
securing screw
Gas cone
6-mm hex screw

Corona discharge
needle
PTFE tubing
11. Clean and sharpen the tip of the needle with the lapping film, then wipe
it clean with a methanol-saturated tissue. Replace the needle if it is
deformed or otherwise damaged.
12. Reinstall the needle with the tip pointing toward the sample cone apex.

13. Close the source enclosure door, and fasten the door’s securing clips.
14. Refit the probe, and reconnect the LC line.
15. Refit the middle panel section.
16. Reconnect the front panel gas and electrical connections.
Cleaning the APCI probe tip

The APcI Probe tip should be cleaned when a buffer build-up is detected on
the probe tip, or when the signal intensity weakens.
To clean the APcI Probe tip:

1. Shut off the liquid flow.
2. Click , or choose Gas on the Gas menu, to start nitrogen flowing.

3. Adjust the nitrogen flow to approximately 650 L/h, as indicated by the
tune page desolvation gas meter.
4. Set the APcI probe heater temperature to 650 °C, and press Enter.
5. Click Operate, and wait 10 minutes with the APcI probe heater at
650 °C. This will remove any chemical contamination from the probe tip.
Replacing the source enclosure and probe adjustment flange

O-rings
Warning: To ensure the integrity of the source exhaust system, the
O-rings listed below must be renewed at intervals of no greater
than one year.
The following O-rings must be renewed at intervals of no greater than one

year:
• Source enclosure door O-ring.
• Source enclosure door glass O-ring.
• Pumping block O-ring.

• Probe adjustment flange O-ring.
• APPI enclosure side flange O-ring (if applicable; refer to the Waters
Micromass APPI/IonSabre Source Operator’s Guide).
Tip: To complete this procedure, you will be required to perform a pressure
test on the source, as described in the Waters Source Pressure Test Unit
Operator’s Guide.
To remove the source enclosure and probe adjustment flange O-rings:

1. Remove the probe from the source (see page 6-8, step 1 to step 9).
Warning: The source can be hot; to avoid burns, take great care
while working with this component.
2. Unfasten the source enclosure door’s securing clips and open the door
(see the figure “Probe and source connections” on page 6-10).
Warning: The source components can be contaminated with

biohazardous and/or toxic materials. Always wear nitrile
gloves while performing this procedure.
3. Use a large flat-bladed screwdriver to remove the three source enclosure

securing screws (see the figure “Source enclosure” on page 6-12).
Tip: To avoid damage, do not apply any force to the source enclosure door
when removing the source enclosure from the instrument’s pumping
block.
4. Remove the source enclosure from the instrument’s pumping block.
Tip: To avoid damage, do not use a metal tool to remove any of the
O-rings.

5. Carefully remove the source enclosure door O-ring from the source
enclosure.

Source enclosure door glass O-ring
Door glass retaining clip
Source enclosure door O-ring
6. Use a hex (Allen) key to remove the four bolts securing the source
enclosure door glass retaining clips to the source enclosure door.
7. Remove the four source enclosure door glass retaining clips from the
source enclosure door.
8. Remove the source enclosure door glass from the source enclosure door.
9. Carefully remove the source enclosure door glass O-ring from the source
enclosure door.
10. Carefully remove the probe adjustment flange O-ring from the probe
adjustment flange.
Probe adjustment flange O-ring
Probe adjustment
flange O-ring

11. Carefully remove the pumping block O-ring from the pumping block (see
the figure “Exploded diagram” on page 6-14).
Warning: The O-rings can be contaminated with

biohazardous and/or toxic materials. Ensure that they are
correctly disposed of according to local environmental
regulations.
12. Dispose of the O-rings in accordance with local environmental

regulations.
To fit the replacement source enclosure and probe adjustment flange

O-rings:
1. Ensure that all the grooves for the O-rings are free from dirt and hairs.
Tip: If contamination is present, use an appropriate solvent, applied to a
lint-free cloth, to carefully clean the grooves.
2. Fit the new pumping block O-ring to the pumping block.
3. Fit the new probe adjustment flange O-ring to the probe adjustment
flange.
4. Fit the new source enclosure door glass O-ring to the source enclosure
door.
5. Fit the source enclosure door glass to the source enclosure door.
6. Fit the four source enclosure door glass retaining clips to the source
enclosure door.
7. Use a hex (Allen) key to fit and tighten the four bolts securing the source
enclosure door glass retaining clips to the source enclosure door.
Tip: Ensure that the source enclosure door O-ring tail is correctly located
in its groove when fitting the O-ring to the source enclosure door.
8. Fit a new source enclosure door O-ring to the source enclosure door.
9. Fit the source enclosure to the pumping block.
Tip: The securing screws must each be sequentially tightened a small
amount until they are all fully tight; this ensures that the source
enclosure is uniformly seated on the pumping block.
10. Fit and tighten the three source enclosure securing screws.

11. Close the source enclosure door and fasten the securing clips.
12. Connect the Probe electrical connection at the instrument’s front panel
(see the figure “Probe and source connections” on page 6-10).
13. Connect the PTFE tubing to the Desolvation gas connection at the
instrument’s front panel.
14. Install the ESI or APCI probe, as required (see page 2-5 and page 2-13).
15. Start up the instrument (see page 2-2).
Warning: To confirm the integrity of the source exhaust

system, you must perform a pressure test on the source, as
described in the Waters Source Pressure Test Unit Operator’s
Guide.
16. Perform a pressure test on the source.
Emptying the nitrogen exhaust waste bottle

Warning: The waste liquid in the nitrogen exhaust waste bottle
comprises LC solvents and analytes. Always wear nitrile gloves while
handling the nitrogen exhaust waste bottle, and ensure that the waste
liquid is correctly disposed of according to local environmental
regulations.
The nitrogen exhaust waste bottle in the nitrogen exhaust line must be
emptied before it is completely full.

Nitrogen exhaust waste bottle
To laboratory
exhaust port
From instrument
exhaust connection
To empty the nitrogen exhaust waste bottle:

1. In the MassLynx™ Tune window, click Press for Standby and confirm
that the adjacent instrument status indicator shows red.
2. In the MassLynx Tune window, click , to stop the nitrogen flow.

3. Disconnect the instrument exhaust and laboratory exhaust system lines
from the nitrogen exhaust waste bottle.
4. Dispose of the waste liquid in accordance with local environmental
regulations.
5. Connect the instrument exhaust and laboratory exhaust system lines to
the nitrogen exhaust waste bottle.
6. In the MassLynx Tune window, click , to start the nitrogen flow.

7. In the Source page Gas Flow pane, set Desolvation (L/hr) to 1200.

8. Set Cone (L/hr) to 300.

system, a leak test must be performed.
®
Tip: To avoid damage to the instrument, Snoop (or equivalent) leak
detector liquid must only be used only for the purpose described in the
following step; it must not be used on any other part of the instrument.
9. Use Snoop (or equivalent) leak detector liquid to ensure that there are
no leaks at the instrument exhaust and laboratory exhaust system line
connections.
Replacing parts
Replacing the ion block cartridge heater

If the cartridge heater fails to heat it must be replaced.
Required materials
• 3 mm hex wrench.
• 1.5 mm hex wrench.
• Needle-nose pliers
Procedure
To replace the ion block cartridge heater:

1. Follow the procedure for venting the instrument as described on
page 6-7.
2. Remove the two screws securing the heater cartridge wires to the PEEK
terminal block.
3. Carefully swing the ring tags out of the terminal block.

Ion block heater
Ring Tags PEEK Terminal Block
Cartridge Heaters
4. Loosen the two set screws that secure the heater cartridges in the ion
block, using the 1.5-mm hex wrench.
5. Gently slide the heater cartridges out of the ion block using the
needle-nose pliers.
6. Slide the new heater cartridges into the ion block with the needle-nose
pliers. Secure them with two hex-head set screws and the 1.5-mm hex
wrench.
7. Position the two heater cartridge ring tags onto the PEEK block
terminals with the bent portion of their shafts extending into the
pumping block
8. Tighten the two terminal block screws with a flat-blade screwdriver.
9. Refit the ion block cover plate, and secure with the four hex screws.
10. Close the source enclosure door and fasten the door’s securing clips.
11. Install the probe.

Replacing the ESI probe stainless steel capillary
The stainless steel sample capillary on the ESI probe must be replaced if it
clogs and cannot be cleared, or if it becomes contaminated or damaged.
ESI probe sample capillary
Stainless Steel
Capillary
Probe
Tip
Required materials
• 1/4-inch (6-mm) wrench
• 5/16-inch wrench
• 7/16-inch wrench
• Loupe (magnifying glass)
• Needle-nose pliers
Procedure
Caution: All work done on the probe should be carried out on a clean
work bench.
To replace the stainless steel capillary:

1. Switch the instrument into standby and remove the probe from the
source.

2. Remove the two end-cover retaining screws on the ESI probe with the
flat-blade screwdriver.
3. Loosen the set screw on the LC PEEK union with the 1.5-mm hex
wrench, and remove the probe’s end-cover.
4. Unscrew the probe tip with the 1/4-inch (6-mm) wrench, and remove it.
5. Remove the LC union with the 5/16-inch and 7/16-inch wrenches.
6. Remove the capillary from the coupling nut with the 7/16-inch wrench.
Discard the capillary and the PTFE liner and ferrule assembly.
7. Remove the conductive sleeve from the inner bore of the probe assembly
fitting.
8. Slide a new ferrule onto the liner tube with the needle-nose pliers.
9. Slide the coupling nut onto the capillary, followed by the PTFE liner
tube and ferrule.
10. Connect a piece of 0.007-inch PEEK tubing with finger-tight nut and
ferrule into the opposite side of the LC union, setting the capillary’s
depth.
11. Press the capillary into the union until it seats, and tighten the adapter
nut to the LC union until it is snug, but not tight.
12. Pull on the capillary gently, testing to ensure it stays in place.
13. Remove the PEEK tubing from the union.
14. Slide the conductive sleeve onto the capillary, then feed the capillary
through the probe.
15. Attach the coupling nut to the probe, and gently tighten it with the
7/16-inch wrench.
16. Replace the probe tip, and screw down until a 0.5-mm length of the
capillary protrudes from its end. Use the loupe, provided in the startup
kit, to check the length of capillary that protrudes from the probe tip.
Caution: Check for leaks carefully! Leakage can destroy a probe.
17. Reconnect the LC line, turn on the fluid flow, and check the probe for
liquid leaks.

18. If a leak is found, disassemble the probe and tighten the fittings at the
LC union.
19. Attach the nebulizer gas connection and turn on the nitrogen, by
clicking on the toolbar (or choose Gas from the Gas menu) on the
tune page.
20. Check the probe tip for nitrogen leaks. If a leak is found, replace the
probe tip assembly and its O-ring.
21. Refit the probe end-cover, and secure it with the two slotted screws.
Tighten the set screw to clamp the LC union in place.
Replacing the ESI probe tip

The ESI probe tip must be replaced if the following problems occur:
• A blockage occurs in the internal metal sheathing through which the
stainless steel capillary passes.
• The threads sustain damage.
Replace the O-ring if gas leaks from the O-ring.
Required materials
• 1/4-inch (6-mm) open-end wrench
Procedure
work bench.
To replace the ESI probe tip:

source.
2. Unscrew and remove the probe tip with the 1/4-inch (6-mm) wrench.
3. Install the new probe tip, and screw down until 0.5 mm of the capillary
protrudes from the end. Use the loupe (provided in the startup kit) to
check the capillary position.

Replacing the APcI probe heater
The APcI probe heater must be replaced if it fails to heat.
Required materials
1 -m hex wrench or flat-blade screwdriver, depending on the type of
screw that secures the probe tip.
Procedure
work bench.
To replace the probe heater:

source.
2. Loosen the two set screws at the base of the probe tip assembly, and
slide the probe tip off.
3. Separate the heater from the probe body, pulling it parallel to the axis of
the probe.
4. Carefully install the new heater onto the probe. Take care not to damage
the fused silica capillary.
5. Refit the probe tip assembly, and secure it with the two set screws.
Replacing the APcI fused silica capillary and filter pad

Replace the fused silica capillary and/or filter pad when a decreased signal
intensity and increased back pressure is noted.
Required materials
• 5/16-inch open-end wrench
• 7/16-inch open-end wrenches (2-off)
• Ceramic capillary cutter (from the tools kit)
• Butane lighter or match

• Lint-free paper towels
Spare parts
• GVF004 ferrules (2-off)
• Fused silica
• Filter pad
Procedure
work bench.
To replace the fused silica capillary and filter pad:

source.
2. Slide the probe tip and heater assembly off the probe.
3. Using the flat-blade screwdriver, remove the two slotted screws.
4. Using the 1.5-mm hex wrench, loosen the two set screws that retain the
LC filter, then remove the probe end.
5. Remove the filter cartridge from the adapter nut with one 5/16-inch and
one 7/16-inch wrench. If the ferrule remains inside the cartridge, remove
it.
6. Separate the two halves of the filter cartridge with two 7/16-inch
wrenches.
7. Remove the old filter pad and replace it with a new one.
8. Unscrew the adapter nut from the probe with a 5/16-inch wrench.
Discard the fused silica capillary.
9. Using the ceramic capillary cutter, cut a new length of fused silica
161.5 mm long. Cut the capillary squarely. Examine new cuts for
squareness with a loupe.

10. Remove approximately 20 mm of polyamide coating from the capillary
end with a flame, then clean it with a methanol-saturated tissue.
11. Slide a GVF004 ferrule, followed by the adapter nut and another
GVF004 ferrule, onto the capillary.
12. Position the filter with the flow direction arrow pointing toward the
adapter nut, then connect the adapter nut to the filter. Make sure the
capillary seats squarely against the filter cartridge interior.
Caution: Overtightening the adapter nut can damage the

capillary.
13. Tighten the adapter nut until the capillary is snug in the fitting, then
gently pull on the capillary to ensure it stays in place
14. Feed the sample capillary through the probe, and gently tighten the
probe adapter nut with the 5/16-inch wrench. The capillary must
protrude 1.0 mm from the nebulizing tube.
15. Refit the probe end-cover and retaining screws.
16. Tighten the set screws in the probe end cover with the 1.5-mm hex
wrench to clamp the filter in place.
17. Refit the probe tip and heater assembly.

7 Troubleshooting
This chapter describes how to troubleshoot the Quattro micro API with
the help of recommended troubleshooting procedures.
Contents
Topic Page
Spare parts 7-2
System troubleshooting 7-3
Component hardware troubleshooting 7-4
High noise levels in MRM analyses 7-13
Electronic noise 7-14
Contacting Waters 7-15
7-1
Spare parts
Parts not included in this document are not recommended for replacement by
the customer.
Safety and handling

When troubleshooting the Quattro micro API, keep the following safety
considerations in mind.
Warning: To avoid the possibility of electric shock,

• never disconnect an electrical assembly while power is applied to the
Quattro micro API. Once power is turned off, wait approximately
10 seconds before disconnecting an assembly.
• To avoid the possibility of electric shock, do not remove the
instrument panels. There are no user-serviceable items inside.
Warning: To prevent injury, always observe good laboratory practices

when handling solvents, changing tubing, or operating Quattro micro
API. Know the physical and chemical properties of the solvents used.
Refer to the Material Safety Data Sheets for the solvents in use.
Caution: To prevent circuit damage due to static charges, do not touch

integrated circuit chips or other components that do not specifically
require manual adjustment.
7-2 Troubleshooting
System troubleshooting
There are a few basic steps for performing system troubleshooting:
• Examine the system, checking the simple things first. Is something
obvious causing the problem (for example, is the instrument and its
cables improperly connected, is there any leakage of fluid, vacuum or
gas?)
• Compare current system operation with the way the system operated
before the problem started. To help identify normal operating
conditions:
– Record a map of the LC system (tubing and electrical connections).
– Keep a daily log.
– Run test samples regularly. Check the instrument performance
with known samples, preferably the ones used for instrument
acceptance.
This illustrates the importance of keeping track of system
parameters and the performance during normal operation.
Troubleshooting is easier if the typical conditions when the system
is operating correctly are known.
For example, are the system tuning parameters similar to those
when a test species was previously run? Are the lens settings
required for optimum sensitivity higher than those previously
obtained? If extreme values have to be used to achieve good results,
this implies that some part of the system requires attention.
• Methodically check and eliminate possible causes to identify the system
parameter that is atypical.
• Refer to the troubleshooting information in the following sections. These
tables enable possible causes of a symptom to be identified and
suggested corrective actions to be taken.
If it is determined that there is a problem related to another system
component (for example HPLC, autosampler, UV detector) refer to the
appropriate operator’s manual.
System troubleshooting 7-3

Component hardware troubleshooting
The following tables provide suggestions for resolving hardware problems.
No peaks on the tune page (no ion beam)

No peaks on the tune page (no ion beam)
Possible cause Corrective action

Operating parameters Optimize parameters. Refer to page 2-12.
(Capillary/Corona, Cone, Once a beam has been obtained, ensure
Extractor, RF Lens, Ion Energy, that all lenses affect the beam in a
Gas Nitrogen and Heaters) on sensible manner.
the tune page are improperly
set.
Cables are not properly Check that all necessary cables have been
connected. correctly attached to the source and probe.
Instrument is not in the operate Put the instrument into operate by
mode. clicking Operate.
When in the operate mode, this icon is
green and the Operate LED on the front
panel is also green.
Communication failure. Re-initialize by going to the tune page and
selecting Options. Reboot the embedded
PC.
No sample is present. Check that sample is loaded correctly in
the autosampler or in the syringe pump
syringe.
Isolation valve is closed. Open the isolation valve.
The source components are Clean the source components. Refer to
dirty. page 6-7.
Insufficient nitrogen flow. Check that the nitrogen pressure is 6 to
7 bar (90 to 100 psi) and the gas flow rate
on the tune page is >100 L/h.
The desolvation and probe heaters shut
off when the nitrogen flow rate falls below
50 L/h.
7-4 Troubleshooting
No peaks on the tune page (no ion beam) (Continued)

No LC flow. Check solvent flow from the autosampler
or syringe pump.
Fluid leak in the HPLC system. Replace the APcI fused silica capillary.
Refer to page 6-33.
Source components have been Check that the source and probe voltage
incorrectly assembled. readbacks vary with the tune page
settings.
If any of these voltages are absent,
disassemble and correctly reassemble the
source and hexapole lens assemblies.
Refer to page 6-7 and page 6-17.
Blocked ESI or APcI capillary Replace capillary.
Refer to page 6-30 and page 6-33.
Unsteady or low intensity peaks (ion beam)

Unsteady or low intensity peaks (ion beam)

Poor nebulization. Check that the source and desolvation
temperature and gas flow settings are suitable
for the flow rate.
Liquid inside the source enclosure is an
indication that the temperature is too low.
The nitrogen pressure should be from 6 to 7 bar
(90 to 100 psi) and desolvation nitrogen flow
rate should be greater than 100 L/h.
Check the stability of the nitrogen flow (good
quality 2-stage regulator).
Problem with sample Troubleshoot the autosampler.
delivery (autosampler, Check the syringe in the syringe pump for
syringe pump or HPLC leaks and grounding.
system).
Check for sufficient sample in the vials.
Look for pressure variation on injection.

Unsteady or low intensity peaks (ion beam) (Continued)

Fluid leak in the HPLC Check for leaks in the HPLC system and
system. correct.
Source components require Clean source components. Refer to page 6-7.
cleaning.
Lens settings wrong or Check all settings are correct.
atypical. Check readbacks are sensible.
Check that all lens parameters affect the beam.
Cone or collision cell Set the voltage ramp off.
voltage ramp is on.
ESI or APcI capillary is Check that the probe position is correct.
not properly installed. Check that the ESI probe stainless steel
capillary protrudes 0.5 mm as described on
page 6-30.
Check that APcI capillary height is set at 1.0
mm. Refer to page 6-33.
Corona pin is not correctly Check the alignment as described on page 6-19.
aligned.
CID gas pressure is wrong. Infuse sample and optimize the gas pressure.
Check the CID gas regulator is set to 0.5 bar,
and is not leaking.
Collision cell parameters Check Entrance, Exit and Collision are
incorrect. optimized, with sensible readbacks.
Analyser and multiplier Ensure Multiplier is 650 V. Check the ion
parameters incorrect. energy and resolution parameters are set
correctly for the acquisition.
7-6 Troubleshooting
Unusually high LC back pressure
Unusually high LC back pressure

Blockage in the capillary or Remove the probe from the source, and
injection loop due to increase the solvent flow to 500 µL/min to
particulate matter from the clear the blockage.
sample.
APcI probe filter pad is Replace the filter pad. Refer to page 6-33.
blocked.
Tubing from LC system is Remove the finger-tight nut and tubing
blocked. from the back of the probe.
If the back pressure remains high, replace
the tubing.
The ESI stainless steel sample Replace the capillary, see page 6-30.
capillary inside the probe is
blocked.
The ESI capillary is not fully Remove and disassemble the probe and
seated in the LC union or the reseat the capillary correctly in the union.
APcI capillary is not properly
seated in the filter.
Unusually low LC back pressure

Unusually low LC back pressure

Leaking connector. Check all fittings and tighten if necessary.
Problem with LC solvent Troubleshoot the LC system.
delivery.
Broken flow cell in UV Replace the flow cell.
detector.

Insufficient vacuum
-4
Any reading greater than 5 × 10 mbar on the Pirani gauge, when CID gas is
off.
Insufficient vacuum

Leaking ion block O-rings. Disassemble source and check condition of
ion block O-rings.
Refer to page 6-7.
Roughing pump not operating Gas ballast the rotary pump to return
correctly. accumulated oil from the oil mist filter.
Check vacuum pump oil. If the oil is dirty,
flush the pump with clean oil, then fill the
pump with oil.
Repeat if necessary.
Leak in vacuum backing line. Check vacuum hose for cracks or vacuum
leaks.
Restriction in vacuum pump Check exhaust line for restrictions.
exhaust tubing.
Turbo pump not operating Check turbo pump speed on the Diagnostics
properly. tune page.
7-8 Troubleshooting
Leaking nitrogen
Warning: To confirm the integrity of the source exhaust system, if
any of the O-rings identified on page 6-30 are renewed, you must
perform a source pressure test as described in the Waters Source
Pressure Test Unit Operator’s Guide.
Hissing sound or solvent smell.
Leaking nitrogen

Poor seal around the source Visually inspect the source enclosure
enclosure. sealing surfaces for imperfections or nicks.
Also, check the condition of the
encapsulated O-rings.
Vacuum oil accumulated in the exhaust tubing

Vacuum oil accumulated in the exhaust tubing

Oil mist filter needs Replace oil mist filter element and odor
replacement. filter.
Source heater and desolvation heater not heating

Source heater and desolvation heater not heating

Source heater has failed. Check readback. Replace heater if
necessary.
Main system PCB fuse failed. Check Desolvation Temp readback. Call
Waters Technical Service if wrong.

APcI heater not heating
APcI heater not heating

If desolvation heater is OK Replace the APcI heater. Refer to page 6-33.
when in ESI mode, then the
APcI heater may need to be
replaced.
Roughing pump fuse fails

Roughing pump fuse fails

Oil mist filter element is Replace oil mist filter element and odor
saturated. filter.
Vacuum oil may also be Replace the fuse.
accumulating in exhaust
tubing.
System needs to be ballasted. Ballast the pump for 20 to 30 minutes.
Refer to page 6-4.
The AC line voltage is less The AC line voltage to the instrument must
than 208 V AC be checked by a qualified electrician.
Vacuum pump oil is very Change vacuum pump oil. Refer to
dirty. page 6-6.
7-10 Troubleshooting
Ion mode fault
Drop-down menu options are grayed out, or instrument spontaneously
switches probe type.
Ion mode fault

One, or both, of the probe Remove probe cover, free the contact pin,
contact pins jammed inside and ensure that both pins and associated
the probe and are not making springs move freely within the bushing.
contact with probe support
plate.
Failure to recognize one particular probe type

Failure to recognize one particular probe type

Problem with the probe. Remove and try another probe of the same
type.
Check that the Source Recognition ID
voltage on the Diagnostics page is <2 V for
ESI, and >2 V for APcI.
Ripple
Peaks and baseline appear to vary cyclically in intensity.
Ripple

Erratic LC solvent flow. Troubleshoot the LC system.
Poor nebulization due to Adjust the temperature and gas flow
incorrect temperature and gas settings. Liquid in the source enclosure is
flow settings. an indication that the temperature is too
low.

Ripple (Continued)

Vibration from the rotary Check for, and eliminate, excessive bench
pumps or even other top and instrument vibration.
equipment in the same
building.
Loss of communication with instrument

Loss of communication with instrument

Instrument to MassLynx host Reset the workstation and reboot the
communication failed. embedded PC from the front panel using a
short length of PEEK tubing to engage the
reset switch, see the figure “Front panel” on
page 1-12.
Wait 3 minutes for the audible signal
indicating the embedded PC has booted
from Quattro micro API before starting
MassLynx.
IEEE communication errors
IEEE communication errors

Instruments powered up in Power down the system components and
the wrong sequence. start up the system components in the
correct order:
1. Workstation
2. Quattro micro API
3. Inlet modules
Wait 3 minutes for the audible signal
indicating the embedded PC has booted
from Quattro micro API before starting
MassLynx.
Wrong IEEE address, or Check system IEEE settings and enter the
conflicting address. correct addresses.
Faulty IEEE cable in IEEE Systematically replace IEEE cables until
chain. the problem cable is located.
Network cables confused with Ensure that the network cable for the
site network. instrument is connected to the correct
network card in the PC.
Ensure that the network card with the
BNC connector is configured to the site
network.
High noise levels in MRM analyses

The background noise in MRM analysis can be either electronic or chemical.
To distinguish between the two:
1. Start an acquisition.
2. During the acquisition, set Ion Energy 1 and Ion Energy 2 fully negative
on the tune page.
A significant decrease in signal when the ion energies are set negative,
implies that the major contribution to the overall noise is chemical.
Any residual noise is electronic.
High noise levels in MRM analyses 7-13

Chemical noise
Chemical noise

High background due to carry-over Repeat injections of 10% formic acid
after tuning with strong and/or isopropanol.
concentrations.
Contaminated injector. (Signal Repeat injections of 10% formic acid
changes upon injection of mobile and/or isopropanol.
phase).
Contaminated tubing. Replace tubing.
Contaminated probe. Flush with methanol at 0.5 mL/min
until the background level falls.
Replace the ESI stainless steel
capillary, see page 6-30.
Replace the APcI fused silica capillary,
see page 6-33.
Contaminated HPLC system. Infuse mobile phase from the solvent
reservoir using a syringe pump.
Compare MRM background
levels.Check purity of solvents.
Replace if necessary.Ensure all
solvents are HPLC grade.
Contaminated glassware. Ensure glassware is not cleaned with
commercial surfactants in the cleaning
process.
Electronic noise
Corrective action
Check that the valleys of peak-peak noise, when ion energies are fully
negative, just touch the baseline. Increase Ion Counting Threshold to suit.
Contacting Waters
Many problems with the Quattro micro API can be easily corrected by the
user. However, if this is not the case, it is necessary to contact Waters.
When contacting Waters, have the following information available:
• Nature of the symptom.
• Quattro micro API serial number.
• Details of flow rate, mobile phases and sample concentrations.
• Details of gas cell operating pressure.
• Tune page settings.
• Software version update reference.
Contacting Waters 7-15

8 Reference Information
Contents
Topic Page
Overview 8-2
Editing a reference file 8-2
Positive ion 8-3
Negative ion 8-6
8-1
Overview
Calibration reference files consist of two columns of numbers separated by any
number of spaces or TAB characters. The first column contains the reference
peak masses and the second column contains the reference peak intensities.
The reference files listed in this chapter have all ion intensities set to 100%.
Actual ion intensities are not, of course, all 100%, but the calibration software
does not take account of the ion intensities and this is a convenient way to
store the reference files in the required format. However, if required, realistic
intensity values can be entered to improve the appearance of the reference
spectra.
Most samples can be purchased from the Sigma chemical company. To order,
contact Sigma at http://www.sigma.sial.com. This site contains a list of
worldwide Sigma offices, many with local toll-free numbers.
Editing a reference file

Calibration reference files can be created or edited using any Windows text
editor.
To read the currently selected reference file into the Notepad text editor:
Press , or select Reference File from the Calibration, Edit menu.
To save the reference file after editing either:

Select Save from the Notepad File menu to save the file under the
current name.
Or:
Select Save as from the Notepad File menu to save as a new reference
file with a new name.
Textual information or comments can be stored in the reference file. Lines
which are textual information or comments must start with the semi-colon (;)
character.
8-2 Reference Information

Positive ion
Ref. file Chemical name Molecular

m/z Uses
name [sigma code #] mass
UBQ Bovine Ubiquitin 8564.85 650 to 1500 General
[U6253]
HBA Human α globin [H753] 15126.36 700 to 1500 Hb analysis
SOD Superoxide dismutase 15591.35 900 to 1500 Hb (internal
[S2515] calibration)
HBB Human β globin [H7379] 15867.22 800 to 1500 Hb analysis
MYO Horse heart myoglobin 16951.48 700 to 1600 General
[M1882]
PEGH10 Polyethylene glycol + 80 to 1000 ES+ and
00 ammonium acetate APcI+
mixture calibration
PEG 200+400+600+1000
PEGH20 Polyethylene glycol + 80 to 2000 ES+
00 ammonium acetate calibration
mixture
PEG
200+400+600+1000+145
0
NAICS Sodium iodide/cesium 20 to 4000 General,
iodide mixture ES+
calibration
NAIRB Sodium iodide/rubidium 20 to 4000 ES+
iodide mixture calibration
Positive ion 8-3

Horse heart myoglobin
Reference File: MYO.REF
Molecular Weight: 16951.48
Charge Calculated Charge Calculated Charge Calculated

state m/z value state m/z value state m/z value
28
+ 606.419 21
+ 808.222 13
+ 1304.969
616.177 20+ 848.583 12+ 1413.633
27+ 628.841 19+ 893.192 11+ 1542.053
26
+ 652.989 18
+ 942.758 10
+ 1696.158
25
+ 679.068 17
+ 998.155 9
+ 1884.508
24+ 707.320 16+ 1060.477 8+ 2119.945
23+ 738.030 15+ 1131.108 7+ 2422.651
22
+ 771.531 14
+ 1211.829
Polyethylene glycol
PEG + NH4+
Reference Files: PEGH1000.REF, PEGH2000.REF
Calculated m/z value

63.04 459.28 855.52 1251.75 1647.99
107.07 503.31 899.54 1295.78 1692.01
151.10 547.33 943.57 1339.80 1736.04
195.12 591.36 987.60 1383.83 1780.07
239.15 635.39 1031.62 1427.86 1824.09
283.18 679.41 1075.65 1471.88 1868.12
327.20 723.44 1119.67 1515.91 1912.15
371.23 767.46 1163.70 1559.94 1956.17
415.25 811.49 1207.73 1603.96 2000.20

Sodium iodide and caesium iodide mixture
Reference File: NAICS.REF
Calculated m/z Value

22.9898 772.4610 1671.8264 2571.1918 3470.5572
132.9054 922.3552 1821.7206 2721.0861 3620.4515
172.8840 1072.2494 1971.6149 2870.9803 3770.3457
322.7782 1222.1437 2121.5091 3020.8745 3920.2400
472.6725 1372.0379 2271.4033 3170.7688
622.5667 1521.9321 2421.2976 3320.6630
Sodium iodide and rubidium iodide

Reference File: NAIRB.REF
Calculated m/z Value

22.9898 772.4610 1671.8264 2571.1918 3470.5572
84.9118 922.3552 1671.8264 2571.1918 3620.4515
172.8840 1072.2494 1821.7206 2721.0861 3770.3457
322.7782 1222.1437 1971.6149 2870.9803 3920.2400
472.6725 1372.0379 2121.5091 3020.8745
622.5667 1521.9321 2271.4033 3170.7688
Positive ion 8-5

Negative ion
Ref. file Chemical name Molecular

m/z Uses
name [sigma code #] mass
MYONEG Horse heart myoglobin 16951.48 700 to 2400 General
[M1882]
SUGNEG Sugar mixture of: 100 to 1500 Low mass
maltose [M5885] range
raffinose [R0250]
maltotetraose [M8253]
corn syrup [M3639]
NAINEG Sodium Iodide/Caesium 200 to 3900 ES-
Iodide (or Rubidium calibration
Iodide) mixture
The purpose of the rubidium iodide is to obtain a peak at m/z 85 (85Rb+) with
an intensity of about 10% of the base peak at m/z 173. Rubidium iodide has
the advantage that no rubidium clusters are formed which may complicate the
85 87
spectrum. Note that rubidium has two isotopes ( Rb and Rb) in the ratio
2.59:1, giving peaks at m/z 85 and 87.
Use reference file NAIRB.REF.
Sodium iodide solution for negative ion ElectroSpray

Either of the above solutions is suitable for calibration in negative ion mode.
In both cases the first negative reference peak appears at m/z 127 (I-) and the
remaining peaks are due to NaI clusters.
Use reference file NAINEG.REF.

A Safety Advisories
Waters instruments display hazard symbols designed to alert you to the

hidden dangers of operating and maintaining the instruments. Their
corresponding user guides also include the hazard symbols, with
accompanying text statements describing the hazards and telling you
how to avoid them. This appendix presents all the safety symbols and
statements that apply to the entire line of Waters products.
Contents
Topic Page
Warning symbols A-2
Caution symbol A-5
Warnings that apply to all Waters instruments A-6
Electrical and handling symbols A-11
A-1
Warning symbols
Warning symbols alert you to the risk of death, injury, or seriously adverse
physiological reactions associated with an instrument’s use or misuse. Heed
all warnings when you install, repair, and operate Waters instruments.
Waters assumes no liability for the failure of those who install, repair, or
operate its instruments to comply with any safety precaution.
Task-specific hazard warnings

The following warning symbols alert you to risks that can arise when you
operate or maintain an instrument or instrument component. Such risks
include burn injuries, electric shocks, ultraviolet radiation exposures, and
others.
When the following symbols appear in a manual’s narratives or procedures,
their accompanying text identifies the specific risk and explains how to avoid
it.
Warning: (General risk of danger. When this symbol appears on an

instrument, consult the instrument’s user documentation for important
safety-related information before you use the instrument.)
Warning: (Risk of burn injury from contacting hot surfaces.)
Warning: (Risk of electric shock.)
Warning: (Risk of fire.)
Warning: (Risk of sharp-point puncture injury.)
Warning: (Risk of hand crush injury.)
Warning: (Risk of exposure to ultraviolet radiation.)
Warning: (Risk of contacting corrosive substances.)
Warning: (Risk of exposure to a toxic substance.)
Warning: (Risk of personal exposure to laser radiation.)
A-2 Safety Advisories

Warning: (Risk of exposure to biological agents that can pose a serious
health threat.)
Warning: (Risk of tipping.)
Warning: (Risk of explosion.)
Warning: (Risk of eye injury.)
Specific warnings
The following warnings can appear in the user manuals of particular
instruments and on labels affixed to them or their component parts.
Burst warning
This warning applies to Waters instruments fitted with nonmetallic tubing.
Warning: Pressurized nonmetallic, or polymer, tubing can burst.

Observe these precautions when working around such tubing:
• Wear eye protection.
• Extinguish all nearby flames.
• Do not use tubing that is, or has been, stressed or kinked.
• Do not expose nonmetallic tubing to incompatible compounds like
tetrahydrofuran (THF) and nitric or sulfuric acids.
• Be aware that some compounds, like methylene chloride and
dimethyl sulfoxide, can cause nonmetallic tubing to swell, which
significantly reduces the pressure at which the tubing can rupture.
Warning symbols A-3

Mass spectrometer flammable solvents warning
This warning applies to instruments operated with flammable solvents.
Warning: Where significant quantities of flammable solvents are

involved, a continuous flow of nitrogen into the ion source is required to
prevent possible ignition in that enclosed space.
Ensure that the nitrogen supply pressure never falls below 690 kPa
(6.9 bar, 100 psi) during an analysis in which flammable solvents are
used. Also ensure a gas-fail connection is connected to the LC system so
that the LC solvent flow stops if the nitrogen supply fails.
Mass spectrometer shock hazard

This warning applies to all Waters mass spectrometers.
Warning: To avoid electric shock, do not remove the mass spectrometer’s

protective panels. The components they cover are not user-serviceable.
This warning applies to certain instruments when they are in Operate mode.
Warning: High voltages can be present at certain external surfaces of

the mass spectrometer when the instrument is in Operate mode. To
avoid non-lethal electric shock, make sure the instrument is in Standby
mode before touching areas marked with this high voltage warning
symbol.

Biohazard warning
This warning applies to Waters instruments that can be used to process
material that might contain biohazards: substances that contain biological
agents capable of producing harmful effects in humans.
Warning: Waters instruments and software can be used to analyze or

process potentially infectious human-sourced products, inactivated
microorganisms, and other biological materials. To avoid infection with
these agents, assume that all biological fluids are infectious, observe
Good Laboratory Practices, and consult your organization’s biohazard
safety representative regarding their proper use and handling. Specific
precautions appear in the latest edition of the US National Institutes of
Health (NIH) publication, Biosafety in Microbiological and Biomedical
Laboratories (BMBL).
Chemical hazard warning

This warning applies to Waters instruments that can process corrosive, toxic,
flammable, or other types of hazardous material.
Warning: Waters instruments can be used to analyze or

process potentially hazardous substances. To avoid injury
with any of these materials, familiarize yourself with the
materials and their hazards, observe Good Laboratory
Practices (GLP), and consult your organization’s safety
representative regarding proper use and handling.
Guidelines are provided in the latest edition of the National
Research Council's publication, Prudent Practices in the
Laboratory: Handling and Disposal of Chemicals.
Caution symbol
The caution symbol signifies that an instrument’s use or misuse can damage
the instrument or compromise a sample’s integrity. The following symbol and
its associated statement are typical of the kind that alert you to the risk of
damaging the instrument or sample.
Caution: To avoid damage, do not use abrasives or solvents to clean the

instrument’s case.
Caution symbol A-5

Warnings that apply to all Waters instruments
When operating this device, follow standard quality control procedures and
the equipment guidelines in this section.
Attention: Changes or modifications to this unit not expressly approved by the

party responsible for compliance could void the user’s authority to operate the
equipment.
Important: Toute modification sur cette unité n’ayant pas été expressément
approuvée par l’autorité responsable de la conformité à la réglementation peut
annuler le droit de l’utilisateur à exploiter l’équipement.
Achtung: Jedwede Änderungen oder Modifikationen an dem Gerät ohne die

ausdrückliche Genehmigung der für die ordnungsgemäße Funktionstüchtigkeit
verantwortlichen Personen kann zum Entzug der Bedienungsbefugnis des
Systems führen.
Avvertenza: qualsiasi modifica o alterazione apportata a questa unità e non

espressamente autorizzata dai responsabili per la conformità fa decadere il
diritto all'utilizzo dell'apparecchiatura da parte dell'utente.
Atencion: cualquier cambio o modificación efectuado en esta unidad que no

haya sido expresamente aprobado por la parte responsable del cumplimiento
puede anular la autorización del usuario para utilizar el equipo.
注意：未經有關法規認證部門允許對本設備進行的改變或修改,可能會使使用者喪失操作該設
備的權利。
注意：未经有关法规认证部门明确允许对本设备进行的改变或改装，可能会使使用者丧失操
作该设备的合法性。
주의: 규정 준수를 책임지는 당사자의 명백한 승인 없이 이 장치를 개조 또는 변경할 경우,

이 장치를 운용할 수 있는 사용자 권한의 효력을 상실할 수 있습니다.
注意：規制機関から明確な承認を受けずに本装置の変更や改造を行うと、本装置のユー
ザーとしての承認が無効になる可能性があります。

Warning: Use caution when working with any polymer tubing under pressure:
• Always wear eye protection when near pressurized polymer tubing.
• Extinguish all nearby flames.
• Do not use tubing that has been severely stressed or kinked.
• Do not use nonmetallic tubing with tetrahydrofuran (THF) or concentrated
nitric or sulfuric acids.
• Be aware that methylene chloride and dimethyl sulfoxide cause nonmetallic
tubing to swell, which greatly reduces the rupture pressure of the tubing.
Attention: Manipulez les tubes en polymère sous pression avec precaution:
• Portez systématiquement des lunettes de protection lorsque vous vous
trouvez à proximité de tubes en polymère pressurisés.
• Eteignez toute flamme se trouvant à proximité de l’instrument.
• Evitez d'utiliser des tubes sévèrement déformés ou endommagés.
• Evitez d'utiliser des tubes non métalliques avec du tétrahydrofurane (THF)
ou de l'acide sulfurique ou nitrique concentré.
• Sachez que le chlorure de méthylène et le diméthylesulfoxyde entraînent le
gonflement des tuyaux non métalliques, ce qui réduit considérablement leur
pression de rupture.
Vorsicht: Bei der Arbeit mit Polymerschläuchen unter Druck ist besondere
Vorsicht angebracht:
• In der Nähe von unter Druck stehenden Polymerschläuchen stets
Schutzbrille tragen.
• Alle offenen Flammen in der Nähe löschen.
• Keine Schläuche verwenden, die stark geknickt oder überbeansprucht sind.
• Nichtmetallische Schläuche nicht für Tetrahydrofuran (THF) oder
konzentrierte Salpeter- oder Schwefelsäure verwenden.
• Durch Methylenchlorid und Dimethylsulfoxid können nichtmetallische
Schläuche quellen; dadurch wird der Berstdruck des Schlauches erheblich
reduziert.

Attenzione: fare attenzione quando si utilizzano tubi in materiale polimerico
sotto pressione:
• Indossare sempre occhiali da lavoro protettivi nei pressi di tubi di polimero
pressurizzati.
• Spegnere tutte le fiamme vive nell'ambiente circostante.
• Non utilizzare tubi eccessivamente logorati o piegati.
• Non utilizzare tubi non metallici con tetraidrofurano (THF) o acido solforico
o nitrico concentrati.
• Tenere presente che il cloruro di metilene e il dimetilsolfossido provocano
rigonfiamenti nei tubi non metallici, riducendo notevolmente la pressione di
rottura dei tubi stessi.
Advertencia: se recomienda precaución cuando se trabaje con tubos de
polímero sometidos a presión:
• El usuario deberá protegerse siempre los ojos cuando trabaje cerca de tubos
de polímero sometidos a presión.
• Si hubiera alguna llama las proximidades.
• No se debe trabajar con tubos que se hayan doblado o sometido a altas
presiones.
• Es necesario utilizar tubos de metal cuando se trabaje con tetrahidrofurano
(THF) o ácidos nítrico o sulfúrico concentrados.
• Hay que tener en cuenta que el cloruro de metileno y el sulfóxido de dimetilo
dilatan los tubos no metálicos, lo que reduce la presión de ruptura de los
tubos.
警告：當在有壓力的情況下使用聚合物管線時，小心注意以下幾點。
• 當接近有壓力的聚合物管線時一定要戴防護眼鏡。
• 熄滅附近所有的火焰。
• 不要使用已經被壓癟或嚴重彎曲管線。
• 不要在非金屬管線中使用四氫呋喃或濃硝酸或濃硫酸。
• 要了解使用二氯甲烷及二甲基亞楓會導致非金屬管線膨脹，大大降低管線的耐壓能力。

警告：当有压力的情况下使用管线时，小心注意以下几点：
• 当接近有压力的聚合物管线时一定要戴防护眼镜。
• 熄灭附近所有的火焰。
• 不要使用已经被压瘪或严重弯曲的管线。
• 不要在非金属管线中使用四氢呋喃或浓硝酸或浓硫酸。
• 要了解使用二氯甲烷及二甲基亚枫会导致非金属管线膨胀，大大降低管线的耐压能力。
경고: 가압 폴리머 튜브로 작업할 경우에는 주의하십시오.

• 가압 폴리머 튜브 근처에서는 항상 보호 안경을 착용하십시오.
• 근처의 화기를 모두 끄십시오.
• 심하게 변형되거나 꼬인 튜브는 사용하지 마십시오.
• 비금속(Nonmetallic) 튜브를 테트라히드로푸란(Tetrahydrofuran: THF) 또는
농축 질산 또는 황산과 함께 사용하지 마십시오.
• 염화 메틸렌(Methylene chloride) 및 디메틸술폭시드(Dimethyl sulfoxide)는
비금속 튜브를 부풀려 튜브의 파열 압력을 크게 감소시킬 수 있으므로 유의하십시오.
警告：圧力のかかったポリマーチューブを扱うときは、注意してください。
• 加圧されたポリマーチューブの付近では、必ず保護メガネを着用してください。
• 近くにある火を消してください。
• 著しく変形した、または折れ曲がったチューブは使用しないでください。
• 非金属チューブには、テトラヒドロフラン(THF)や高濃度の硝酸または硫酸などを流
さないでください。
• 塩化メチレンやジメチルスルホキシドは、非金属チューブの膨張を引き起こす場合が
あり、その場合、チューブは極めて低い圧力で破裂します。

Warning: The user shall be made aware that if the equipment is used in a
manner not specified by the manufacturer, the protection provided by the
equipment may be impaired.
Attention: L’utilisateur doit être informé que si le matériel est utilisé d’une
façon non spécifiée par le fabricant, la protection assurée par le matériel risque
d’être défectueuses.
Vorsicht: Der Benutzer wird darauf aufmerksam gemacht, dass bei

unsachgemäßer Verwenddung des Gerätes die eingebauten
Sicherheitseinrichtungen unter Umständen nicht ordnungsgemäß
funktionieren.
Attenzione: si rende noto all'utente che l'eventuale utilizzo

dell'apparecchiatura secondo modalità non previste dal produttore può
compromettere la protezione offerta dall'apparecchiatura.
Advertencia: el usuario deberá saber que si el equipo se utiliza de forma

distinta a la especificada por el fabricante, las medidas de protección del equipo
podrían ser insuficientes.
警告：使用者必須非常清楚如果設備不是按照製造廠商指定的方式使用，那麼該設備所提供
的保護將被消弱。
警告：使用者必须非常清楚如果设备不是按照制造厂商指定的方式使用，那么该设备所提供
的保护将被削弱。
경고: 제조업체가 명시하지 않은 방식으로 장비를 사용할 경우 장비가 제공하는 보호 수단이

제대로 작동하지 않을 수 있다는 점을 사용자에게 반드시 인식시켜야 합니다.
警告：ユーザーは、製造元により指定されていない方法で機器を使用すると、機器が提供
している保証が無効になる可能性があることに注意して下さい。

Electrical and handling symbols
Electrical symbols
These can appear in instrument user manuals and on the instrument’s front
or rear panels.
Electrical power on
Electrical power off
Standby
Direct current
Alternating current
Protective conductor terminal
Frame, or chassis, terminal
Fuse
Recycle symbol: Do not dispose in municipal waste.
Electrical and handling symbols A-11

Handling symbols
These handling symbols and their associated text can appear on labels affixed
to the outer packaging of Waters instrument and component shipments.
Keep upright!
Keep dry!
Fragile!
Use no hooks!

B Materials of Construction and
Compatible Solvents

system, you must address any safety issues raised by the
contents of this Appendix.
Contents
Topic Page
Preventing contamination B-2
Items exposed to solvent B-2
Solvents used to prepare mobile phases B-3
B-1
Preventing contamination
For information on preventing contamination, refer to Controlling
Contamination in LC/MS Systems (part number 715001307). Visit
www.waters.com.
Items exposed to solvent

The items that appear in the following table can be exposed to solvent. You
must evaluate the safety issues if the solvents used in your application differ
from the solvents normally used with these items. See page B-3 for details
about the most common ingredients used to prepare mobile phases.]
Items exposed to solvent
Item Material
Corona discharge pin mounting PEEK (Polyetheretherketone)
contact
Gas exhaust port Aluminium
Gas tubes FEP (Fluorinated ethylene
propylene)
Ion block Stainless steel
Ion block support PEEK (Polyetheretherketone)
Isolation valve Gold-plated aluminium/bronze
O-rings Viton or
®
PTFE (Polytetrafluoroethylene)-
encapsulated Viton
Probe adjuster bellows PTFE
(Polytetrafluoroethylene)/Viton
Probe adjuster assembly Anodized aluminium, glass filled
acetal, and stainless steel
Probe shaft PEEK (Polyetheretherketone)
Push-in gas fittings Nickel/brass
Source enclosure Alochromed aluminium
B-2 Materials of Construction and Compatible Solvents

Items exposed to solvent (Continued)
Item Material
Source enclosure view port Toughened plate glass
Waste bottle Polypropylene
Solvents used to prepare mobile phases

The following lists the most common ingredients used to prepare mobile
phases for reverse-phase LC/MS (API):
• Water
• Methanol
• Acetonitrile
• Formic acid (<0.1%)
• Acetic acid (<0.1%)
• Trifluoroacetic acid (<0.1%)
• Ammonium acetate (<10 mM)
• Ammonium formate (<10 mM)
These solvents are not expected to cause any problems with the materials
identified on page B-2.
Solvents used to prepare mobile phases B-3

B-4 Materials of Construction and Compatible Solvents
C Environmental Specifications
.
Contents
Topic Page
Access C-2
Location C-2
Dimensions and clearances C-4
Weights C-6
Operating temperature C-7
Operating humidity C-7
Shipping and storage temperature C-7
C-1
Access
Doors through which the instrument is to be moved should be a minimum of
24 inches (0.6 m) wide. Elevators and corridors must be wide enough to allow
corners to be negotiated. Special arrangements may be required if access to
the laboratory is via a staircase.
Location
Quattro micro API

The Quattro micro API may be installed upon any flat bench top.
The instrument should not be placed close to heavy machinery (compressors,
generators, etc.) which causes excessive floor vibration.
The instrument should not be placed within a RF field of greater than
specified in IEC 801-3 severity level 2.
Possible sources of RF emission include RF-linked alarm systems or LANs,
portable telephones and hand held transmitters.
The instrument must be positioned away from strong magnetic fields such as
those generated by NMR systems and magnetic sector mass spectrometers.
The instrument is shipped with a 8 feet (2.5 m) power cable that must be
plugged into the rear of the chassis. The power ON/OFF switch is located on
the front panel.
It is recommended that the Quattro micro API be positioned at the right-hand
end of a bench to allow extra room for access to the electronics.
Vacuum pump
The rotary pump should be installed beneath, or behind, the Quattro micro
API, within 5 feet (1.5 m) of the rear of the chassis. The rotary pump is fitted
with a 6.5 feet (2 m) power cable which plugs into the rear of the instrument’s
chassis. It is recommended that the rotary pump is elevated 6 to 8 inches(15 to
20 cm) above the floor to aid in routine maintenance, such as changing pump
oil.
If the rotary pump is sited under the instrument bench, an access slot may
need to be drilled in the bench top for routing of vacuum tubing from the
C-2 Environmental Specifications

instrument (see the figure “Horizontal clearances” on page C-4 for
specifications). The access slot for the vacuum tubes must be within the
footprint of the instrument and must be 31.3 inches (800 mm) from the
proposed position of the instrument’s front face.
Data system
The data system must be located within 16 feet (5 m) of the mass spectrometer
to allow connection of the communication cables. The data system power
cables are approximately 6.5 feet (2 m) in length. A typical layout for the data
system is shown in the figure “Data system” on page C-5.
LC system
Be sure to allow adequate space to the left of the instrument for the HPLC
system. Refer to the associated user manuals for individual space
requirements.
Location C-3
Dimensions and clearances
Horizontal clearances

Data system
Vertical clearances
Dimensions and clearances C-5

Ideally, the following minimum clearances should be allowed for service
access and ventilation:
• 8 inches(20 cm) on the right side [Note: 20 inches (0.5 m) is required for
periodic service of the main PCB, refer to the figure “Vertical clearances”
on page C-5.]
• 11 inches (28 cm) on the top
It is acceptable for the instrument to be placed with its back to a wall with a
minimum of 4 inches (10 cm) clearance between the wall and the back of the
instrument for ventilation.
Height
23 in. (572 mm)
Length
34.6 in. (880 mm)
Width
15.4 in.(390 mm).
Weights
The bench must be able to support the total weight of the system:
Instrument: 253 lb (115 kg)
Data system (computer, monitor and optional printer): 110 lb (50 kg)
Total Weight: 363 lb (165 kg)
Lifting and carrying

As the instrument weight is 253 lb (115 kg), it is recommended that either
suitable lifting equipment, or an appropriate number of physically able
personnel are available to lift a unit of this weight, positioned for equal
distribution of the load. Waters personnel are not permitted to manually lift
the instrument without suitable assistance.

As a general guide before lifting, lowering or moving the instrument:
• Assess the risk of injury
• Take action to eliminate risk
If some risk still exists:
• Plan the operation in advance and in conjunction with our engineer
when he/she arrives on site.
• Use trained personnel where necessary
• Adhere to appropriate country and/or company regulations.
Operating temperature
The ambient temperature range for instrument operation is 15 to 30°C (59 to
86°F), with short-term variation (1.5 h) not exceeding 2°C (4°F). The ambient
temperature range for rotary pump operation is 15 to 40°C (59 to 104°F).
The maximum overall heat dissipation into the room is 3.0 kW. This figure
does not take into account ancillary equipment such as LC systems.
Air-conditioning may have to be installed, or upgraded, to accommodate the
additional heat load into the room.
Operating humidity
The relative humidity range for instrument operation is 20 to 80%. It is
recommended that the instrument be situated in a draft-free position in an
air-conditioned laboratory free from excessive amounts of dust.
Shipping and storage temperature

0 to 40°C (32 to 104°F).
Operating temperature C-7

D Electrical Specifications
Contents
Topic Page
Installation D-2
Electrical safety D-4
D-1
Installation
It is recommended that the electrical installation in the laboratory include a
wall-mounted emergency isolator switch of the correct rating, which is
protected by a correctly-rated circuit breaker. The isolator should be clearly
marked “Mass Spectrometer” and ideally positioned with clear access at all
times for emergency switch-off, without risk of slipping or tripping. Ideally,
the isolator should be capable of being locked in the OFF position to prevent
unauthorized personnel from switching the instrument on. The Quattro micro
API must have an earthed power supply that satisfies the voltage and current
specifications for that country, detailed below:
Quattro micro API (including rotary vacuum pump)

Voltage and Frequency 230 V, 50/60 Hz (+8%, -14%)
Minimum Current 13 A
Maximum Current 16 A
Power Consumption 2.0 kW
Power Cord Supplied 8 ft. (2.5 m), fitted with country plugs as shown
below.
Canada:
The user must supply a fused supply, rated at 15 A.
UK:
The cable is fused at 13 A.
Japan:
A single phase, 3-wire 200 V 5% phase/neutral supply, rated between 13
and16 A must be supplied. Additionally, a transformer must be ordered
from Waters to boost the input voltage.
Australia and New Zealand:
The equipment has been designed in compliance with the international safety
standard IEC1010-1 (EN61010-1). To be fully effective, the building
installation must comply with AS 3000: Electrical Installations for
Australia/New Zealand.
Rest of World:
The user must supply a fused supply, rated between 13 and 16 A.
D-2 Electrical Specifications

Plugs supplied
USA EUROPE UK
NEMA L6-15P, 15 A 2-pin Europlug 3-pin UK plug, fused at
conforming to CEE7 13 A, BS1633
standards
For countries with a 60 Hz AC. supply (such as the USA), if the supply voltage
is less than 208 V AC., it is recommended that a step-up transformer is used
to boost the voltage to 208 to 230 V AC.
The mains supply must have at least a ±10% brown out capability, i.e. 10% for
0.3 seconds and transient ≤20ms to half mains voltage.
Appropriate sockets to mate with the plugs shown must be provided.
If a plug is required other than that shown, the user must supply the plug and
the appropriate socket.
The instrument is shipped with a 8 feet(2.5 m) power cable that must be
plugged into the rear of the chassis. The Quattro micro API power ON/OFF
switch is located on the front panel.
Line frequency
50 Hz, 47 to 53 Hz.
60 Hz, 57 to 63 Hz.
Installation D-3
Fuse rating
10 A, 250 V AC.
Electrical safety
The Waters Quattro micro API conforms to European standard
EN61010-1:2001, Safety requirements for electrical equipment for
measurement, control, and laboratory use - Part 1: General requirements.
The instrument is categorized as Pollution Category 1 and Installation
Category 2.
D-4 Electrical Specifications

E Performance Specifications
ElectroSpray positive ion

The measured signal/noise ratio obtained from the chromatogram monitoring
the transition m/z 609 to m/z 195 on injection of 10 pg (16 fmol) of reserpine
will be >20:1.
10 µL of a 1 pg/µL reserpine solution in 50/50 acetonitrile/water (no additives)
will be injected at a flow rate of 200 µL/min, in MRM mode, 1 second dwell,
span 0 Da. The resolution of the ion at m/z 609 will be ≤1 Da peak width at
half height (MS1 and MS2).
ElectroSpray negative ion

The signal/noise ratio measured on the [M-H]- peak at m/z 503 from a sample
consumption of 10 ng raffinose will be >100:1.
A solution of 5 ng/µL Raffinose in 50/50 acetonitrile/water (no additives) will
be introduced at a flow rate of 10 µL/min and the summation of two 6 second
scans over the mass range m/z 100 to 600 represents a total sample
consumption of 10 ng.
APcI positive ion

Measured signal/noise ratio obtained from the mass chromatogram of m/z 609
in SIR mode on direct injection of 10 pg (16 fmol) reserpine (10 µL injection of
a 1 pg/µL solution) at a flow rate of 1 mL/min will be >10:1.
ElectroSpray positive ion E-1

E-2 Performance Specifications
Index
A Constant neutral loss 1-11
Acquisition status window 4-8 construction materials B-1
Analog data channels 4-22 Contact closure 1-17
Analog input channels 1-16 contamination, preventing B-2
APcI Probe heater 1-13 Corona discharge needle 6-19
APcI probe tip 6-22
Atmospheric Pressure Chemical D
Ionization 1-5 Data acquisition - stopping 4-16
audience and purpose viii Data system 1-3
Automated quantification 4-6 Daughter ion 1-6
Automatic Calibration Check 5-9 Desolvation gas 1-12
AutoTune 3-10 Divert/injection valve 1-14
Drug metabolite 1-10
B
Baseline 3-17 E
biohazard warning A-5 EC authorized representative xi
burst warning A-3 electrical symbols A-11
ElectroSpray ionization 1-4
C ElectroSpray operation - preparing for
Caesium iodide 8-5 2-7
Calibration - checking 5-18 Embedded PC 1-3
Calibration - failure 5-21 Environmental analysis 1-10
Calibration - removing 5-9 equipment guidelines viii, A-6
Calibration - saving 5-24 ESI / APcI multi-way connector 1-13
Calibration - starting 5-18 ESI heater 1-13
Calibration parameters 5-11 ESI Probe 2-5
Capillary electrophoresis interlock Extraction cone 6-15
1-17
caution symbol A-5 F
Centroid data 4-12 flammable solvents A-4
chemical hazard warning A-5 Forensic science 1-10
Chromatogram - real-time update 4-9 FractionLynx control 1-17
CID gas valve 1-13 Front panel 1-12
Collision energy ramp 3-16 Full Scan function 4-24
compatible solvents B-1 Function List Editor 4-16
Cone gas 1-12 Function List Editor toolbar 4-19
Cone voltage ramp 3-15
Index-1
G Negative ion 8-6
Grid 3-10
O
H Operate LED 1-15
handling symbols A-12
Herbicides 1-10 P
Hexapole assembly 6-18 Parent ion 1-8
PC link 1-17
I Peptides 1-7
Instrument data thresholds 4-10 Pesticides 1-10
intended use ix Pharmacokinetic studies 1-10
Intensity 3-10 Pirani gauge 1-3
Ion block 6-13 Precursor ion 1-8
Ion Counting Threshold 4-12 preventing contamination B-2
Product ion 1-6
M Profile data 4-11
Mass flow controllers 1-12 Profile data - spike removal 4-14
Mass Measure parameters 5-12 purpose and audience viii
mass spectrometer shock hazard A-4
MassLynx 1-4 R
materials of construction B-1 Readbacks 3-17
MaxEnt 4-11 Rear panel connections 1-16
mobile phases, solvents used in Reserpine 1-7, 1-9, 1-10
preparation of B-3 Rotary pump - gas ballasting 6-4
Mode 3-13 Rotary pump oil - changing 6-6
Monitoring acquisitions 4-39 Rotary pump oil - checking 6-5
MRM 1-10 Routine maintenance 6-4
MRM function 4-32 Rubidium iodide 8-5
MS operating modes 1-5
MS to MS/MS switching 4-35 S
MS/MS operating modes 1-6 safety advisories A-1
MS/MS scanning functions 4-30 Sample inlet 1-3
MS/MS to MS switching 4-37 Sample Inlet 1-3
MS1 mode 1-5 Scan Report window 4-8
MS2 mode 1-5 Scan speed compensation calibration
Multiple Reaction Monitoring 1-10 5-4
Mux Interface 1-17 Scanning calibration 5-4
Scope 3-13
N SIR data 4-12
Nebulizer gas 1-12 SIR function 4-28
Index-2
Sodium iodide 8-5 Z
Software 1-4 Zero level 3-17
Solvent delay 4-21
solvents I
compatible B-1
exposure of SQ detector
components to B-2
use in mobile phases B-3
Source enclosure 6-12
Spectrum - real-time update 4-9
Starting 2-2
Static calibration 5-4
Status display 1-15
Survey function 4-33
symbols
caution A-5
electrical A-11
handling A-12
warning A-2
System Manager 4-16
T
Toxicology 1-10
Trace 3-10
Tune page 3-2
Tune Parameter settings - changing
3-18
Tuning 3-2
Turbomolecular pump 1-3
U
UV detector 1-16
V
Vacuum LED 1-15
Vacuum system 1-3
W
warning symbols A-2, A-6
Index-3
Index-4

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Waters Quattro micro

API Mass Spectrometer

Copyright © Waters Corporation 2003−2010

Waters contact information

Contacting medium Information

Considerations specific to the Waters Quattro micro API

Flammable solvents hazard

Warning: To prevent the ignition of accumulated solvent vapors inside

Warning: To avoid burn injuries, avoid touching the source enclosure

Mass spectrometer high temperature hazard

Mass spectrometer in ESI ionization mode

Ion block and cone

ESI probe tip

Warning: To avoid personal contamination with

Warning: To avoid puncture injuries, handle syringes, fused silica lines,

Authorized representative of the European

Australia C-Tick EMC Compliant

Confirms that a manufactured product complies

Consult instructions for use

For in vitro diagnostic use

Audience and purpose

ISM Classification: ISM Group 1 Class A

Waters Corporation (Micromass UK Ltd.)

Contacting Waters ............................................................................................... iii

Safety considerations .......................................................................................... iii

Operating this instrument .............................................................................. viii

ISM classification .................................................................................................. x

EC authorized representative ........................................................................... xi

1 Instrument Description ........................................................................ 1-1

Sample inlet ........................................................................................................ 1-3

Vacuum system .................................................................................................. 1-3

Data system ........................................................................................................ 1-3

MassLynx software ........................................................................................... 1-4

Theory and principles of operation .............................................................. 1-4

Table of Contents xiii

Front panel controls, indicators and connections .................................. 1-12

Rear panel connections ................................................................................. 1-16

2 Routine Procedures ............................................................................... 2-1

Installing the ESI (ElectroSpray) probe ...................................................... 2-5

Setting up the syringe pump .......................................................................... 2-6

Setting up the Quattro micro API ................................................................. 2-7

3 Tuning ....................................................................................................... 3-1

The tune page .................................................................................................... 3-2

Printing tune information .............................................................................. 3-2

Experimental record ........................................................................................ 3-3

Saving and restoring tune parameter settings .......................................... 3-4

Modifying the peak display ............................................................................ 3-5

xiv Table of Contents

AutoTune ........................................................................................................... 3-10

Ion mode ............................................................................................................ 3-13

Scope parameters ............................................................................................ 3-13

Gas controls ...................................................................................................... 3-14

Resetting the zero level ................................................................................. 3-17

Controlling readbacks ................................................................................... 3-17

Changing tune parameter settings ............................................................. 3-18

4 Data Acquisition ..................................................................................... 4-1

Monitoring an acquisition .............................................................................. 4-8

Instrument data thresholds .......................................................................... 4-10

System manager .............................................................................................. 4-16

Stopping an acquisition ................................................................................ 4-16

5 Mass Calibration .................................................................................... 5-1

Overview ............................................................................................................. 5-2

ElectroSpray ...................................................................................................... 5-5

xvi Table of Contents